Wiki FKKT - User contributions [en]

2015-bionano-seminar

2015-03-29T07:26:18Z

EvaKnapič:

= Bionanotehnologija- seminar =
doc. dr. Gregor Gunčar, K2.022

== Seznam seminarjev ==
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Avtor 1'''
| align="center" style="background:#f0f0f0;"|'''Avtor 2'''
| align="center" style="background:#f0f0f0;"|'''Naslov seminarja'''
| align="center" style="background:#f0f0f0;"|'''Datum za oddajo'''
| align="center" style="background:#f0f0f0;"|'''Datum predstavitve'''
| align="center" style="background:#f0f0f0;"|'''Recenzent 1'''
| align="center" style="background:#f0f0f0;"|'''Recenzent 2'''
|-
| Anže Prašnikar||Monika Praznik ||||29.03.||31.03.||Aneja Tuljak||Angelika Vižintin
|-
| Varja Božič||Eva Knapič||Razgradljivi kondomi s protimikrobno zaščito||29.03.||31.03.||Eva Udovič||Maja Grdadolnik
|-
| Belkisa Velagić||Aleksander Benčič||||29.03.||31.03.||Nika Kurinčič||Tjaša Goričan
|-
| Naja Vrankar||Valter Bergant||||31.03.||02.04.||Nataša Žigante||Luka Smole
|-
| Tilen Volčanšek||Veronika Jarc||||31.03.||02.04.||Anže Prašnikar||Jakob Gašper Lavrenčič
|-
| Tanja Lipec||Iza Ogris||||31.03.||02.04.||Varja Božič||Klara Tereza Novoselc
|-
| Katja Lovrin||Mitja Crček||||05.04.||07.04.||Belkisa Velagić||Monika Praznik
|-
| Saša Balažic||Urban Javoršek ||||05.04.||07.04.||Naja Vrankar||Eva Knapič
|-
| Urban Borštnik||Sara Primec||||05.04.||07.04.||Tilen Volčanšek||Aleksander Benčič
|-
| Nives Ahlin||Kim Kos||||12.04.||14.04.||Tanja Lipec||Valter Bergant
|-
| Matic Bevec||Estera Merljak||||12.04.||14.04.||Katja Lovrin||Veronika Jarc
|-
| Vida Špindler||Jernej Pušnik||||12.04.||14.04.||Saša Balažic||Iza Ogris
|-
| Jasmina Sedmak||Maxi Sagmeister||||19.04.||21.04.||Urban Borštnik||Mitja Crček
|-
| Sanja Popović||Benjamin Bajželj||||19.04.||21.04.||Nives Ahlin||Urban Javoršek
|-
| Blaž Komar||Alja Zottel||||19.04.||21.04.||Matic Bevec||Sara Primec
|-
| Blaž Perič||Katarina Uršič||||03.05.||05.05.||Vida Špindler||Kim Kos
|-
| Simon Preložnik||Maja Remškar||||03.05.||05.05.||Jasmina Sedmak||Estera Merljak
|-
| Kaja Javoršek||Tina Gregorič||||03.05.||05.05.||Sanja Popović||Jernej Pušnik
|-
| Damir Hamulić||Anita Kustec||||10.05.||12.05.||Blaž Komar||Maxi Sagmeister
|-
| Janja Fortin||Tina Snoj||||10.05.||12.05.||Blaž Perič||Benjamin Bajželj
|-
| Rajko Vnuk||Mojca Banič||||10.05.||12.05.||Simon Preložnik||Alja Zottel
|-
| Rok Grm||Ajda Rojc||||17.05.||19.05.||Kaja Javoršek||Katarina Uršič
|-
| Kristina Gavranić||Barbara Žužek||||17.05.||19.05.||Damir Hamulić||Maja Remškar
|-
| Urška Mohorič||Griša Prinčič||||17.05.||19.05.||Janja Fortin||Tina Gregorič
|-
| Maja Ramić||Nejc Petrišič||||24.05.||26.05.||Rajko Vnuk||Anita Kustec
|-
| Barbara Jeras||Tamara Marić||||24.05.||26.05.||Rok Grm||Tina Snoj
|-
| Matic Urlep||Samo Zakotnik||||24.05.||26.05.||Kristina Gavranić||Mojca Banič
|-
| Urban Verbič||Angelika Vižintin||||31.05.||02.06.||Urška Mohorič||Ajda Rojc
|-
| Nataša Žigante||Maja Grdadolnik||||31.05.||02.06.||Maja Ramić||Barbara Žužek
|-
| Aneja Tuljak||Tjaša Goričan||||31.05.||02.06.||Barbara Jeras||Griša Prinčič
|-
| Eva Udovič||Luka Smole||||07.06.||09.06.||Matic Urlep||Nejc Petrišič
|-
| Nika Kurinčič||Jakob Gašper Lavrenčič||||07.06.||09.06.||Urban Verbič||Tamara Marić
|-
| Klara Tereza Novoselc||||||07.06.||09.06.||Samo Zakotnik||
|}

== Gradivo za predavanja ==
Gradivo za predavanja najdete v [http://ucilnica.fkkt.uni-lj.si/ spletni učilnici].

==Naloga==
'''Vaša naloga je: '''
Po dva študenta skupaj pripravita projektno nalogo iz področja Bionanotehnologije. Najpomembnejša je originalna ideja za nek izvedljiv projekt.
Predlagana struktura:
* Uvod
* Predstavitev problema, znanstvena izhodišča, cilji
* Izvedba projekta, metodologija, tehnike, materiali, vprašanja, hipoteze
* Literatura

Za pripravo seminarja velja naslednje: 
* Prva stran seminarja naj vsebuje naslov projekta, avtorje, povzetek (od 130 do 160 besed) in grafični povzetek (čez približno pol strani)
* Seminar pripravite v obliki seminarske naloge na ~5 straneh A4 (pisava 12, enojni razmak, 2,5 cm robovi). Zelo pomembno je, da je obseg od 1500 do 2000 besed . Seminarska naloga mora vsebovati najmanj tri slike. Slika mora imeti legendo in v besedilu mora biti na ustreznem mestu sklic na sliko. 
* Seminar oddajte do datuma oddaje, ki je naveden v tabeli v elektronski obliki z uporabo [http://bio.ijs.si/~zajec/poslji/ tega obrazca].
* Vsi seminarji so v elektronski obliki dostopni [http://bio.ijs.si/~zajec/poslji/bioseminar/ tukaj].
* Ustna predstavitev sledi na dan, ki je vpisan v tabeli. Za predstavitev je na voljo 20 minut, predstavitev pa ne sme biti krajša od 15 minut (popust :-)). Nalogo predstavita oba študenta (razdelita si čas). Recenzenti morajo biti na predstavitvi prisotni.
* Predstavitvi sledi razprava. Recenzenti podajo pripombe k projektu in postavijo po dve vprašanji.
* Na dan predstavitve morate docentu še pred predstavitvijo oddati končno verzijo seminarja v enem izvodu, elektronsko verzijo seminarja in predstavitev pa oddati na strežnik na dan predstavitve do polnoči.

==Imena datotek==
Prosim vas, da vse datoteke poimenujete po naslednjem receptu:
* 19_nano_Priimek1_Priimek2.doc(x) za seminar, npr. 19_nano_Craik_Venter.docx
* 19_nano_Priimek1_Priimek2.ppt(x) za prezentacijo, npr. 19_nano_Craik_Venter.pptx

==Ocenjevanje seminarjev==
Recenzenti ocenijo seminar tako, da izpolnijo [https://docs.google.com/forms/d/1WdCXoXo1zkRrVlLKIcEV1z_MyhavU-3ERBm9n2oiawI/viewform recenzentsko poročilo] na spletu. Recenzentsko poročilo morate oddati najkasneje do predstavitve seminarja.

== Mnenje o predstavitvi ==
Vsak posameznik '''mora''' oceniti seminar, tako da odda svoje [https://docs.google.com/forms/d/1ToLPn78T9W3G6Hm5hV0mLseFYghiLQMlRPGb0J5zft8/viewform mnenje] najkasneje v sedmih dneh po predstavitvi. Kdor na seminarju ni bil prisoten, mnenja '''ne sme''' oddati.

==Urejanje spletnih strani na wikiju==
Wiki so razvili zato, da lahko spletne vsebine ureja vsakdo. Ukazi so preprosti, dokler si ne zamislite česa prav posebnega. Vseeno pa je Word v primerjavi z wikijem pravo čudežno orodje... Če imate težave z oblikovanjem besedila, si preberite poglavje o urejanju wiki-strani na Wikipediji ([http://en.wikipedia.org/wiki/Help:Editing tule] v angleščini in [http://sl.wikipedia.org/wiki/Wikipedija:Urejanje_strani tu] v slovenščini). Pomaga tudi, če pogledate, kako je zapisana kakšna stran, ki se vam zdi v redu: kliknite na zavihek 'Uredite stran' in si poglejte, kako so vpisane povezave, kako nov odstavek in podobno. ''Na koncu seveda pod oknom za urejanje kliknite na 'Prekliči'.''

==Citiranje virov==
Citiranje je možno po več shemah, važno je, da se držite ene same. V seminarskih nalogah in diplomskih nalogah FKKT uprabljajte shemo citiranja, ki je pobarvana zeleno.
Temeljno načelo je, da je treba vir navesti na tak način, da ga je mogoče nedvoumno poiskati.
Za citate v naravoslovju je najpogostejše citiranje po pravilniku ISO 690. [http://www.zveza-zotks.si/gzm/dokumenti/literatura.html Pravila], ki upoštevajo omenjeni standard, so pripravili pri ZTKS. Sicer pa ima vsaka revija lahko svoj način citiranja, ki ga je treba pri pisanju članka upoštevati. 
'''Citiranje knjig:''' 
Priimek, I. ''Naslov''. Kraj: Založba, letnica. 
Priimek, I. ''Naslov: podnaslov''. Izdaja. Kraj: Založba, letnica. Zbirka, številka. ISBN. 

Boyer, R. ''Temelji biokemije''. Ljubljana: Študentska založba, 2005. 
Glick BR in Pasternak JJ. ''Molecular biotechnology: principles and applications of recombinant DNA''. 3. izdaja. Washington: ASM Press, 2003. ISBN 1-55581-269-4. 
Če so avtorji trije, je beseda in med drugim in tretjim avtorjem. Če so avtorji več kot trije, napišemo samo prvega in dopišemo ''et al''. (in drugi, po latinsko). Vse, kar je latinsko, pišemo poševno (npr. tudi imena rastlin in živali, pojme ''in vivo'', ''in vitro'' ipd.).

'''Citiranje člankov:''' 
Priimek, I. Naslov. ''Naslov revije'', letnica, letnik, številka, strani. 
Lartigue, C., Glass, J. I., Alperovich, N., Pieper, R., Parmar, P. P., Hutchison III, C. A., Smith, H. O. in Venter, J. C.
Genome transplantation in bacteria: changing one species to another. ''Science'', 2007, 317, str. 632-638.

Alternativni način citiranja (predvsem v družboslovju) je po pravilih APA, kjer članke citirajo takole: 
Priimek, I. (letnica, številka). Naslov. Naslov revije, strani. 
Lartigue C. ''et al.'' (2007, 317) Genome transplantation in bacteria: changing one species to another. ''Science'', 632-638.

Revija Science uporablja skrajšani zapis: 
C. Lartigue ''et al''. Science 317, 632 (2007) 

V diplomah na FKKT je treba navesti vire tako, da izpišete tudi naslov citiranega dela in strani od-do (ne samo začetne). Navesti morate tudi vse avtorje dela, razen v primeru, ko jih je 10 ali več. Takrat navedite le prvih devet, za ostale pa uporabite okrajšavo in sod. (in sodelavci). Pred zadnjim avtorjem naj bo vedno besedica "in".

'''Citiranje spletnih virov:''' 
Priimek, I. ''Naslov dokumenta''. Izdaja. Kraj: Založnik, letnica. Datum zadnjega popravljanja. [Datum citiranja.] spletni naslov 
strangeguitars. ''On the brink of artificial life''. 6. 10. 2007. [citirano 13. 11. 2007] http://www.metafilter.com/65331/On-the-brink-of-artificial-life 
Navedemo čim več podatkov; pogosto vseh iz pravila ne boste našli.

An improved zinc-finger nuclease architecture for highly specific genome editing

2015-01-11T18:42:44Z

EvaKnapič:

(Eva Knapič)

Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J.[http://www.nature.com/nbt/journal/v25/n7/full/nbt1319.html An improved zinc-finger nuclease architecture for highly specific genome editing]. ''Nat Biotechnol''., 2007, 25(7), 778-785.

== Genome editing ==
Genome editing is a type of genetic engineering in which DNA is inserted, removed or replaced from genome using artificially designed nucleases, enzymes that cleave the phosphodiester bonds between nucleotides. The principle of method is based on nuclease cleavage at a desired site of the genome in order to create specific double-stranded break, then put to use the cell’s endogenous mechanisms to repair the induced break by homology-directed repair or non-homologous end joining. There are a few different classes of engineered nucleases that are currently being used: zinc-finger nucleases, transcription activator-like effector nucleases and the CRISPR/Cas system. In this seminar we will focus on zinc finger nucleases. Firstly, we will take a look at basics of zinc-finger nucleases and their mechanism. Then we will discuss improved zinc-finger nuclease architecture for more specific efficiency described by Miller and colleagues in their paper published in Nature Biotechnology in 2007<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>.

== Zinc-finger nuclease ==
Zinc-finger nuclease (ZFN) is an artificially designed endonuclease that can be customised to cleave at sequence specific site on the DNA. The enzyme consists of two domains: DNA-binding domain composed of zinc finger structural motifs and DNA-cleavage domain of restriction enzyme FokI. Domains are connected with short inter-domain linker. This structure combines key qualities of DNA binding specificity, flexibility of zinc-finger motifs and cleavage activity of FokI catalytic domain that is robust but restricted. Furthermore all three elements can be optimised for retargeting and improving efficiency<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref><ref name="ref2">Urnov, F. D.; Rebar, E.J.; Holmes, M. C.; Zhang, H. S.; Gregory, P.D. Genome editing with engineered zinc finger nucleases. ''Nat Rev Genet''., 2010, 11(9), 636-646.</ref>.

=== DNA-binding domain ===
As mentioned above, zinc finger motifs represent DNA-binding domain of ZFN. [http://commons.wikimedia.org/wiki/File:Zinc_finger_rendered.png#mediaviewer/File:Zinc_finger_rendered.png Zinc fingers] are small protein structural motifs distinguished by coordinated zinc ions that stabilise the protein fold. Each zinc-finger recognises 3 base pairs (bp) of DNA. Contacts between the zinc fingers and DNA are made by α-helix that binds in the DNA major groove<ref name="ref3">Zinc finger ; Wikipedia the free encylopedia [cited 2.1.2015]. http://en.wikipedia.org/wiki/Zinc_finger.</ref>. Proteins that contain any number of zinc finger motifs are called zinc-finger proteins (ZFPs). Engineered ZFPs consist of three to six zinc finger motifs thus enabling binding of 9 bp to 18 bp targets. Longer recognition sequence improves specificity and precision of ZFPs. ZFP based DNA-binding domains can be coupled to various effector domains, which then cuts the DNA sequence determined by the ZFP<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref><ref name="ref2">Urnov, F. D.; Rebar, E.J.; Holmes, M. C.; Zhang, H. S.; Gregory, P.D. Genome editing with engineered zinc finger nucleases. ''Nat Rev Genet''., 2010, 11(9), 636-646.</ref>.

There are multiple approaches to designing new ZFPs with binding specificities chosen by user. The simplest and widely used method to generate new zinc finger sets is modular assembly. Candidate ZFPs for target sequence are obtained by determining individual zinc fingers that bind each triplet and assembling them together thus recognising the target sequence<ref name="ref2">Urnov, F. D.; Rebar, E.J.; Holmes, M. C.; Zhang, H. S.; Gregory, P.D. Genome editing with engineered zinc finger nucleases. ''Nat Rev Genet''., 2010, 11(9), 636-646.</ref>. Alternative methods for designing new ZFPs include two-finger modules, oligomerised pool engineering (OPEN) and Context-Dependent Assembly (CoDA). In two-finger modules instead of individual fingers, construct of two-fingers is used for recognising target DNA sequence. Advantage of this approach is better optimisation of finger junctions and speed of finding and assembling suitable zinc fingers, a limitation is extension of initial characterisation of two-finger units <ref name="ref4">Carroll, D. Genome engineering with zinc-finger nucleases. ''Genetics''., 2011, 188(4), 773-782.</ref>. The OPEN system approach is carried out in two steps, firstly appropriate finger pools are recombined to create small combinatorial library, then members of library that efficiently bind to the target site are isolated using bacterial two-hybrid selection method in which binding of a zinc finger domain to its target activates expression of selected marker gene<ref name="ref5">Maeder M. L. et al. Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification. ''Mol Cell''., 2008, 31(2), 294–301.</ref>. CoDA is publicly available platform of reagents and software. With this approach three-finger sets are assembled using N- and C- terminal fingers that have been previously identified in other arrays with common middle finger. CoDA does not treat fingers as independent modules but instead explicitly accounts for context-dependent effects between adjacent fingers, increasing the probability of efficiency <ref name="ref6">Sander J.D. et al. Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA). ''Nat Methods''., 2011, 8(1), 67-69.</ref>. Regardless of which approach is used for designing new ZFPs ''in vitro'', their application in living cells is not always successful. Reason is the complexity of genome that often contains naturally occurring multiple copies of sequence that are either identical or very similar to target sequence and these copies can act as additional targets for ZFNs. Another complication is chromatin structure at target sites that may obstruct cleavage<ref name="ref4">Carroll, D. Genome engineering with zinc-finger nucleases. ''Genetics''., 2011, 188(4), 773-782.</ref>.

=== Inter-domain linker ===
Inter-domain linker connects DNA-binding domain with DNA-cleavage domain. Length of the linker is responsible for the shaping of spacer. Spacer is short sequence between both recognition sites for DNA-binding domains, which enables precise definition of cleavage site. Wilson and colleagues have demonstrated that sites with 5 bp spacer lengths showed the most efficient ZFN mediated gene targeting when the linker is 2 or 4 amino acids and that sites with 3 or 4 bp spacers could not be targeted efficiently. Group also demonstrated that ZFN variants with 5 amino acids linker can efficiently target sites with a 7 bp spacer. However, the ideal linker with corresponding spacer can only be determined by taking into consideration both ZFN activity on the chromosomal target site and ZFN associated toxicity <ref name="ref7">Wilson, K.A.; McEwen, A.E.; Pruett-Miller, S.M.; Zhang, J.; Kildebeck, E.J.;, Porteus, M.H. Expanding the Repertoire of Target Sites for Zinc Finger Nuclease-mediated Genome Modification. Mol Ther Nucleic Acids., 2013, 2, e88.</ref>.

=== DNA-cleavage domain ===
DNA-cleavage domain of ZFN originates from FokI catalytic domain. FokI is type IIS restriction endonuclease consisting of an N-terminal DNA-binding domain and non-specific DNA-cleavage C-terminal domain. In order to cleave DNA two FokI catalytic domains must form dimer. Each subunit contains one catalytic centre that is responsible for cleaving one strand of DNA duplex. This characteristic of FokI catalytic domain is crucial for successful genome editing with ZFN. Dimerization requires two independent binding events, which must occur in correct orientation and spacing between monomers. DNA-cleavage domain is usually fused to the C-terminus of DNA-binding domain, thus two individual ZFNs must bind to opposite strands of DNA. This enables specific targeting of long and potentially unique recognition sites<ref name="ref2">Urnov, F. D.; Rebar, E.J.; Holmes, M. C.; Zhang, H. S.; Gregory, P.D. Genome editing with engineered zinc finger nucleases. ''Nat Rev Genet''., 2010, 11(9), 636-646.</ref>.

== ZFN-mediated genome editing ==
ZFNs create double-stranded breaks (DSBs) in DNA at sequence specific site. DSBs can be repaired by cell’s endogenous mechanisms: homology-directed repair (HDR) or non-homologous end joining (NHEJ). There are few different possible outcomes of DSB repair. If the break is repaired by NHEJ, it can lead to gene disruption, tag ligation or large deletion. Gene disruption occurs when two broken ends are ligated back together that can lead to small insertions or deletions at the break site causing target gene disruption. Tag ligation occurs when adaptor with sticky ends is provided and is ligated into the chromosome producing tagged allele. When two different ZFNs cleave simultaneous same chromosome, entire segment between them is deleted. If the break is repaired by HDR, which occurs in the presence of donor DNA, it can result in gene correction, targeted gene addition or transgene stacking. If donor with small change, for example single bp, is provided, editing of endogenous allele results in gene correction. This enables easier functional genomics studies, modelling of disease caused by point mutation and gene therapy research. If provided donor carries transgene and has specific homology arms, transgene can be efficiently integrated into chromosome at break site, which results in targeted gene addition. Transgene stacking occurs when provided donor carries multiple linked transgenes between the homology arms. With this approach not only gene addition can be done, but also gene correction if DSB is created within or near gene in question and provided donor carries modified gene sequence<ref name="ref2">Urnov, F. D.; Rebar, E.J.; Holmes, M. C.; Zhang, H. S.; Gregory, P.D. Genome editing with engineered zinc finger nucleases. ''Nat Rev Genet''., 2010, 11(9), 636-646.</ref><ref name="ref4">Carroll, D. Genome engineering with zinc-finger nucleases. ''Genetics''., 2011, 188(4), 773-782.</ref><ref name="ref8">Gaj, T.; Gersbach, C.A.; Barbas, C.F. 3rd. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. ''Trends Biotechnol''., 2013, 31(7), 397-405.</ref>.

=== Applications: ===
==== Model organisms ====
ZFNs enable introduction of targeted modification in several model organisms used in common biological researches. Benefits of DSB repaired by cell’s endogenous mechanisms include: simple production of knock-out or knock-in cell lines, permanent and heritable mutation, compatibility with variety of species<ref name="ref8">Gaj, T.; Gersbach, C.A.; Barbas, C.F. 3rd. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. ''Trends Biotechnol''., 2013, 31(7), 397-405.</ref>.
==== Therapeutic application of ZFNs ====
ZFN-mediated gene modification by HDR has been used to correct mutations associated with X-linked severe combined immune deficiency (SCID), haemophilia B, sickle-cell disease and α1-antitrypsin deficiency. ZFN induced gene targeting by NHEJ repair has been shown as potential treatment for HIV/AIDS, either by targeting cellular co-receptors for HIV in primary T cells and hematopoietic stem/progenitor cells (clinical trials) or by targeting proviral HIV DNA<ref name="ref8">Gaj, T.; Gersbach, C.A.; Barbas, C.F. 3rd. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. ''Trends Biotechnol''., 2013, 31(7), 397-405.</ref>.

=== Potential problems ===
If the DNA-binding domain is not specific enough or target site is not unique in genome, ZFN may cleave at undesired location in the genome. Another potential problem is formation of ZFN homodimers that are by products of ZFN heterodimer that can cleave off-target. Besides cleaving off-target, ZFN homodimers mediate toxic effect. Undesired cleavage can lead to unwanted insertions or deletions at additional cleavage sites or vast number of DSBs can cause cell death. This can be avoided by extensive bioinformatics research beforehand, construction of ZFP that recognises longer and consequently rarer target sequences, using suitable linkers and cleavage domains that do not form unwanted homodimers<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref><ref name="ref2">Urnov, F. D.; Rebar, E.J.; Holmes, M. C.; Zhang, H. S.; Gregory, P.D. Genome editing with engineered zinc finger nucleases. ''Nat Rev Genet''., 2010, 11(9), 636-646.</ref>.

== AN IMPROVED ZINC-FINGER NUCLEASE ARCHITECTURE ==
Miller and colleagues present in their paper an improved ZFN architecture for more specific genome targeting. Their aim was to develop FokI cleavage domain variants that function as obligate heterodimers<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>.

=== Development of heterodimeric FokI cleavage domain variants ===
There are few things that need to be put in consideration when altering architecture of FokI DNA-cleavage domain. Firstly, FokI is an enzyme, this means goal is to improve interaction between domains but at the same time, catalytic function of domains must be conserved. Secondly, interaction between FokI DNA-cleavage domains in dimer has very low affinity, which is essential for ensuring cleavage specificity. Lastly, interface of FokI dimer is hydrophilic. Considering all the facts, Miller and colleagues developed strategy that involved step by step approach to modification of dimer interface coupled with direct testing of candidate variants for catalytic activity. Reporter system with green fluorescent protein (eGFP) was used to detect gene correction activity. The system uses HEK293 cell line containing a copy of the eGFP gene that is disrupted by short DNA fragment and has characterised ZFN target sequence [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F2.html (Figure 2a)]. Screening is based on ZFN cleavage of DNA fragment that is corrected by HDR using exogenous donor DNA containing missing eGFP sequence as template. If repair is successful, green fluorescence can be detected by fluorescence-activated cell sorting [http://commons.wikimedia.org/wiki/File:Fluorescence_Assisted_Cell_Sorting_(FACS)_B.jpg#mediaviewer/File:Fluorescence_Assisted_Cell_Sorting_(FACS)_B.jpg (FACS)]<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>. FACS is specialized type of flow cytometry, method for separating heterogeneous mixture of biological cells, which is based upon the specific light scattering at fluorescent wavelength<ref name="ref9">Flow cytometry; Wikipedia the free encylopedia [cited 2.1.2015]. http://en.wikipedia.org/wiki/Flow_cytometry#Fluorescence-activated_cell_sorting_.28FACS.29.</ref>. Results are shown in [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F2.html Figure 2b], left for cells transfected with donor DNA only and right for cells transfected with donor DNA and ZFNs. Step by step modification of interaction surface was carried out in four cycles. Modifications were implemented alternately, which means in each cycle only one partner domain was modified. Mutations were designed to form charge-charge interaction. Each variant was tested for gene correction activity as a heterodimer with unmodified partner ZFN and as a homodimer. More in detailed view of introduced mutation can be found in [http://www.nature.com/nbt/journal/v25/n7/suppinfo/nbt1319_S1.html Supplementary table 1]. In each cycle researchers identified a variant of domain that induced gene correction as a heterodimer and at the same time the domain had reduced activity as a homodimer [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F2.html (Figure 2d)]. Variants with best gene correction rate contained double mutations E490K:I538K and Q486E:I499L (also known as EL:KK). In one domain glutamic acid (E) at position 490 and isoleucine (I) at position 538 were replaced with lysine (K) (E490K:I538K), in partner domain glutamine (Q) at position 486 was replaced with glutamic acid and isoleucine at position 499 was replaced with leucine (L) (Q486E:I499L). As lysine is positively charged amino acid, variants with two additional lysine residues were referred to as ‘+’ and the other variants were referred to as ‘-‘, due to negative charge of introduced glutamic acid. Model of how variants may interact with each other is shown in [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F2.html Figure 2e]. Variants ‘+’ and ‘-‘ exhibited strong preference for heterodimerization and very weak preference for homodimerization when connected with suitable ZFP (L or R ZFP) in GFP reporter system. In [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F2.html Figure 2f] we can see that double mutations heterodimers (marked as L+/R- and L-/R+) have higher efficiency for gene correction than wild-type (wt) dimer (marked as Lwt/Rwt) and homodimers of double mutation variants (marked as L+/R+ and L-/R-) have almost none. Results of GFP reporter system indicate that formation of obligated heterodimer was achieved<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>.

=== Validation of heterodimeric FokI cleavage domain variants ===
==== Efficient endogenous gene editing ====
To confirm that the heterodimer variants preserved catalytic function researchers fused them with ZFPs that target a sequence present in exon 5 of the human gene for interleukin-2 receptor γ-chain (IL2Rγ). Designed ZFNs induced gene editing that introduced novel BsrBI restriction site in IL2Rγ [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F3.html (Figure 3a)]. Restriction enzyme BsrBI is able to cut at such restriction site resulting in additional band on electrophoresis. Results of gene editing efficiency shown in [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F3.html Figure 3b] indicate BsrBI restriction site was successfully inserted in case of ZFN with wt cleavage domain (Lwt/Rwt) and both heterodimeric combinations (L+/R- and L-/R+). Forced homodimers (L+/R+ and L-/R-) yielded no detectable gene editing. These results confirmed obligated heterodimerization of developed variants<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>.

==== Suppression of homodimer function ''in vitro'' ====
Cleavage activity ''in vitro'' was directly measured by radiolabeling of target sequences for ZFNs [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F4.html (Figure 4a)]. Target sequences were then digested by ZFNs with wt cleavage domain and variants of domain with double mutations, both in heterodimeric and homodimeric form. Migration of cleaved and uncleaved products in [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F4.html Figure 4b] indicate that wt cleavage domain (Lwt/Rwt) was active in heterodimeric and homomeric form, while efficient cleavage with double mutation variants (L+/R- and L-/R+) was only observed if domains formed heterodimer. This result once again supports development of variants, which form obligated heterodimer<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>.

==== Reduced levels of genome-wide cleavage ====
The aim of study was to develop more specific ZFNs that do not cleave off-target. Therefore, researchers conducted the next experiment on ZFN specificity of gene modification in mammalian cells. ZFN cleavage was determined by detection of proteins that localise at DNA DSBs. The proteins bound were detected by antibody-mediated technique. Protein that localise at DNA damage site and form foci, is tumour suppressor p53-binding protein 1 (53BP1). Protein 53BP1 was detected by immunofluorescence in target cells, which were transfected with ZFN expression constructs and treated with anti-53BP1 rabbit polyclonal antibodies. Results in [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F5.html Figure 5a] show that signal is weaker in cells transfected with ZFN with double mutations variants compared to ZFN with wt cleavage domain. This indicates cleavage activity was reduced in case of transfection with modified ZFN. Western blot analysis [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F5.html (Figure 5b)] confirmed that both ZFNs were expressed at comparable levels, suggesting that the reduced activity was a result of decreased off-target effect. Alternative damage marker is phosphorylated histone H2AX (γH2AX), which also forms foci at DSBs. Flow cytometry data [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F6.html (Figure 6a)] for target cells transfected with ZFNs and stained with antibodies against γH2AX showed that number of positive cells (cells forming γH2AX foci) was significantly lower for ZFNs with double mutations variants compared to ZFN with wt cleavage domain. In addition gene modification was monitored by Surveyor Nuclease assay, which determines the frequency of the small insertions and deletions (indels) characteristic of unspecific DSB repair by NHEJ. Results [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F6.html (Figure 6b)] showed comparable levels of gene modification in cells treated with ZFNs with wt and double mutations cleavage domains, indicating comparable cellular activities at target locus. These results confirmed that heterodimeric FokI cleavage domain variants retain catalytic activity while exhibiting reduced levels of off-target cleavage<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>.

=== Conclusion ===
In conclusion, Miller and colleagues developed two complementary FokI cleavage domain variants with double mutation, which function as an obligate heterodimer and improve ZFN specificity limiting off-target effect. Researchers predicted that their new architecture of FokI cleavage domain could be beneficial for therapeutic application of ZFNs due to additional mechanism for reducing off-target cleavage. It could also be helpful for less safety-intensive application such as crop engineering, cell-line engineering and construction of disease models. These ZFNs would be useful for gene modification protocols requiring simultaneous cleavage at multiple targets, where it would eliminate unwanted combination of ZFNs<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>.

== Further research ==
In 2011 same research group form Sangamo BioSciences published a [http://www.nature.com/nmeth/journal/v8/n1/full/nmeth.1539.html new paper] in Nature Methods discussing improved obligate heterodimeric architectures. In this study yeast-based selection system was used to cross-examine the dimer interface. A reporter assay was used to isolate FokI with mutations causing cold-sensitivity, phenotype resulting in diminished cleavage activity at lower temperatures but still be active at higher ones. The hypothesis of study was that cold-sensitive mutants would affect critical residues involved in dimerization, because this class of mutations is often associated with incorrect assembly of multisubunit protein complexes<ref name="ref10">Doyon, Y.; Vo, T.D.; Mendel, M.C.; Greenberg, S.G.; Wang, J.; Xia, D.F.; Miller, J.C.; Urnov, F.D.; Gregory, P.D.; Holmes, M.C. Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures. ''Nat Methods''., 2011, 8(1), 74-79.</ref>.

A selection system in ''Saccharomyces cerecisiae'' developed to isolate ZFN mutants with cold-sensitivity phenotype included two independent single-stranded annealing reporter constructs that were integrated into the yeast genome. DNA sequence for reporter genes MEL1 and PHO5 were interrupted by binding site for ZFN homodimer (CCR5-L) [http://www.nature.com/nmeth/journal/v8/n1/fig_tab/nmeth.1539_F1.html (Figure 1a)]. Additionally PHO5 reporter system had incorporated positive selection cassette natMX that enables resistance to antibiotic nourseothricin. MEL1 reporter system contained the URA3 gene for negative selection using 5-fluoroorotic acid, which can be converted into the toxic compound causing cell death and kanMX cassette that enables resistance to antibiotic geneticin. ZFN-induced DSB would result in restoration of reporter gene expression and simultaneous elimination of all positive and negative selection markers. Library of FokI mutants was constructed by error-prone PCR that randomly mutagenized the sequence [http://www.nature.com/nmeth/journal/v8/n1/fig_tab/nmeth.1539_F1.html (Figure 1b)]. ZFN expression was induced at 22 °C, and then cells were collected and incubated in medium with geneticin and nourseothricin. With this step all cells carrying active ZFN constructs were eliminated. Remaining cells were shifted to 37 °C and plated on medium with 5-fluoroorotic acid and colorimetric substrate for Pho5. This step gave them conditionally active ZFNs. Constructs were then isolated and tested at 22 °C, 30 °C and 37 °C to confirm cold-sensitive phenotype [http://www.nature.com/nmeth/journal/v8/n1/fig_tab/nmeth.1539_F1.html (Figure 1c)]; 16 mutants had minimal activity at 22 °C but had restored reporter gene expression at higher temperature. Within isolated mutants previously altered residues that contributed to obligated heterodimerization (glutamine 486 and isoleucine 499 and 538) were found. Their focus was residues asparagine 496 and histidine 537, which face each other in dimerization interface. These two residues were substituted with a pair of oppositely charged amino acids into EL:KK FokI backbone. This substitution could strengthen dimerization in the context of the obligate heterodimer variants. The asparagine 496 was replaced with negatively charged glutamic and aspartic acid in the EL domain, and histidine 537 was replaced with positively charged lysine and arginine in the KK domain. Measured cleavage activity comparted to both wt and EL:KK versions suggested that aspartic acid 496 drove maximal activity in EL monomer, whereas lysine and arginine at position 537 resulted in similar activity improvements. This domains were referred to as ELD:KKK or ELD:KKR. Improved activity of new FokI variants was confirmed by various assays. All results supported superior cleavage activity and retainment of obligate heterodimer function. Researchers concluded that these enhanced FokI domains were portable to many ZFPs, independent of cell type and are a general solution for improved ZFN activity<ref name="ref10">Doyon, Y.; Vo, T.D.; Mendel, M.C.; Greenberg, S.G.; Wang, J.; Xia, D.F.; Miller, J.C.; Urnov, F.D.; Gregory, P.D.; Holmes, M.C. Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures. ''Nat Methods''., 2011, 8(1), 74-79.</ref>.

==References==
<references />

[[SB students resources]]

An improved zinc-finger nuclease architecture for highly specific genome editing

2015-01-11T17:43:39Z

EvaKnapič:

(Eva Knapič)

Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J.[http://www.nature.com/nbt/journal/v25/n7/full/nbt1319.html An improved zinc-finger nuclease architecture for highly specific genome editing]. ''Nat Biotechnol''., 2007, 25(7), 778-785.

== Genome editing ==
Genome editing is a type of genetic engineering in which DNA is inserted, removed or replaced from genome using artificially designed nucleases, enzymes that cleave the phosphodiester bonds between nucleotides. The principle of method is based on nuclease cleavage at a desired site of the genome in order to create specific double-stranded break, then put to use the cell’s endogenous mechanisms to repair the induced break by homology-directed repair or non-homologous end joining. There are a few different classes of engineered nucleases that are currently being used: zinc-finger nucleases, transcription activator-like effector nucleases and the CRISPR/Cas system. In this seminar we will focus on zinc finger nucleases. Firstly, we will take a look at basics of zinc-finger nucleases and their mechanism. Then we will discuss improved zinc-finger nuclease architecture for more specific efficiency described by Miller and colleagues in their paper published in Nature Biotechnology in 2007<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>.

== Zinc-finger nuclease ==
Zinc-finger nuclease (ZFN) is an artificially designed endonuclease that can be customised to cleave at sequence specific site on the DNA. The enzyme consists of two domains: DNA-binding domain composed of zinc finger structural motifs and DNA-cleavage domain of restriction enzyme FokI. Domains are connected with short inter-domain linker. This structure combines key qualities of DNA binding specificity, flexibility of zinc-finger motifs and cleavage activity of FokI catalytic domain that is robust but restricted. Furthermore all three elements can be optimised for retargeting and improving efficiency<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref><ref name="ref2">Urnov, F. D.; Rebar, E.J.; Holmes, M. C.; Zhang, H. S.; Gregory, P.D. Genome editing with engineered zinc finger nucleases. ''Nat Rev Genet''., 2010, 11(9), 636-646.</ref>.

=== DNA-binding domain ===
As mentioned above, zinc finger motifs represent DNA-binding domain of ZFN. [http://commons.wikimedia.org/wiki/File:Zinc_finger_rendered.png#mediaviewer/File:Zinc_finger_rendered.png Zinc fingers] are small protein structural motifs distinguished by coordinated zinc ions that stabilise the protein fold. Each zinc-finger recognises 3 base pairs (bp) of DNA. Contacts between the zinc fingers and DNA are made by α-helix that binds in the DNA major groove<ref name="ref3">Zinc finger ; Wikipedia the free encylopedia [cited 2.1.2015]. http://en.wikipedia.org/wiki/Zinc_finger.</ref>. Proteins that contain any number of zinc finger motifs are called zinc-finger proteins (ZFPs). Engineered ZFPs consist of three to six zinc finger motifs thus enabling binding of 9 bp to 18 bp targets. Longer recognition sequence improves specificity and precision of ZFPs. ZFP based DNA-binding domains can be coupled to various effector domains, which then cuts the DNA sequence determined by the ZFP<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref><ref name="ref2">Urnov, F. D.; Rebar, E.J.; Holmes, M. C.; Zhang, H. S.; Gregory, P.D. Genome editing with engineered zinc finger nucleases. ''Nat Rev Genet''., 2010, 11(9), 636-646.</ref>.

There are multiple approaches to designing new ZFPs with binding specificities chosen by user. The simplest and widely used method to generate new zinc finger sets is modular assembly. Candidate ZFPs for target sequence are obtained by determining individual zinc fingers that bind each triplet and assembling them together thus recognising the target sequence<ref name="ref2">Urnov, F. D.; Rebar, E.J.; Holmes, M. C.; Zhang, H. S.; Gregory, P.D. Genome editing with engineered zinc finger nucleases. ''Nat Rev Genet''., 2010, 11(9), 636-646.</ref>. Alternative methods for designing new ZFPs include two-finger modules, oligomerised pool engineering (OPEN) and Context-Dependent Assembly (CoDA). In two-finger modules instead of individual fingers, construct of two-fingers is used for recognising target DNA sequence. Advantage of this approach is better optimisation of finger junctions and speed of finding and assembling suitable zinc fingers, a limitation is extension of initial characterisation of two-finger units <ref name="ref4">Carroll, D. Genome engineering with zinc-finger nucleases. ''Genetics''., 2011, 188(4), 773-782.</ref>. The OPEN system approach is carried out in two steps, firstly appropriate finger pools are recombined to create small combinatorial library, then members of library that efficiently bind to the target site are isolated using bacterial two-hybrid selection method in which binding of a zinc finger domain to its target activates expression of selected marker gene<ref name="ref5">Maeder M. L. et al. Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification. ''Mol Cell''., 2008, 31(2), 294–301.</ref>. CoDA is publicly available platform of reagents and software. With this approach three-finger sets are assembled using N- and C- terminal fingers that have been previously identified in other arrays with common middle finger. CoDA does not treat fingers as independent modules but instead explicitly accounts for context-dependent effects between adjacent fingers, increasing the probability of efficiency <ref name="ref6">Sander J.D. et al. Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA). ''Nat Methods''., 2011, 8(1), 67-69.</ref>. Regardless of which approach is used for designing new ZFPs ''in vitro'', their application in living cells is not always successful. Reason is the complexity of genome that often contains naturally occurring multiple copies of sequence that are either identical or very similar to target sequence and these copies can act as additional targets for ZFNs. Another complication is chromatin structure at target sites that may obstruct cleavage<ref name="ref4">Carroll, D. Genome engineering with zinc-finger nucleases. ''Genetics''., 2011, 188(4), 773-782.</ref>.

=== Inter-domain linker ===
Inter-domain linker connects DNA-binding domain with DNA-cleavage domain. Length of the linker is responsible for the shaping of spacer. Spacer is short sequence between both recognition sites for DNA-binding domains, which enables precise definition of cleavage site. Wilson and colleagues have demonstrated that sites with 5 bp spacer lengths showed the most efficient ZFN mediated gene targeting when the linker is 2 or 4 amino acids and that sites with 3 or 4 bp spacers could not be targeted efficiently. Group also demonstrated that ZFN variants with 5 amino acids linker can efficiently target sites with a 7 bp spacer. However, the ideal linker with corresponding spacer can only be determined by taking into consideration both ZFN activity on the chromosomal target site and ZFN associated toxicity <ref name="ref7">Wilson, K.A.; McEwen, A.E.; Pruett-Miller, S.M.; Zhang, J.; Kildebeck, E.J.;, Porteus, M.H. Expanding the Repertoire of Target Sites for Zinc Finger Nuclease-mediated Genome Modification. Mol Ther Nucleic Acids., 2013, 2, e88.</ref>.

=== DNA-cleavage domain ===
DNA-cleavage domain of ZFN originates from FokI catalytic domain. FokI is type IIS restriction endonuclease consisting of an N-terminal DNA-binding domain and non-specific DNA-cleavage C-terminal domain. In order to cleave DNA two FokI catalytic domains must form dimer. Each subunit contains one catalytic centre that is responsible for cleaving one strand of DNA duplex. This characteristic of FokI catalytic domain is crucial for successful genome editing with ZFN. Dimerization requires two independent binding events, which must occur in correct orientation and spacing between monomers. DNA-cleavage domain is usually fused to the C-terminus of DNA-binding domain, thus two individual ZFNs must bind to opposite strands of DNA. This enables specific targeting of long and potentially unique recognition sites<ref name="ref2">Urnov, F. D.; Rebar, E.J.; Holmes, M. C.; Zhang, H. S.; Gregory, P.D. Genome editing with engineered zinc finger nucleases. ''Nat Rev Genet''., 2010, 11(9), 636-646.</ref>.

== ZFN-mediated genome editing ==
ZFNs create double-stranded breaks (DSBs) in DNA at sequence specific site. DSBs can be repaired by cell’s endogenous mechanisms: homology-directed repair (HDR) or non-homologous end joining (NHEJ). There are few different possible outcomes of DSB repair. If the break is repaired by NHEJ, it can lead to gene disruption, tag ligation or large deletion. Gene disruption occurs when two broken ends are ligated back together that can lead to small insertions or deletions at the break site causing target gene disruption. Tag ligation occurs when adaptor with sticky ends is provided and is ligated into the chromosome producing tagged allele. When two different ZFNs cleave simultaneous same chromosome, entire segment between them is deleted. If the break is repaired by HDR, which occurs in the presence of donor DNA, it can result in gene correction, targeted gene addition or transgene stacking. If donor with small change, for example single bp, is provided, editing of endogenous allele results in gene correction. This enables easier functional genomics studies, modelling of disease caused by point mutation and gene therapy research. If provided donor carries transgene and has specific homology arms, transgene can be efficiently integrated into chromosome at break site, which results in targeted gene addition. Transgene stacking occurs when provided donor carries multiple linked transgenes between the homology arms. With this approach not only gene addition can be done, but also gene correction if DSB is created within or near gene in question and provided donor carries modified gene sequence<ref name="ref2">Urnov, F. D.; Rebar, E.J.; Holmes, M. C.; Zhang, H. S.; Gregory, P.D. Genome editing with engineered zinc finger nucleases. ''Nat Rev Genet''., 2010, 11(9), 636-646.</ref><ref name="ref4">Carroll, D. Genome engineering with zinc-finger nucleases. ''Genetics''., 2011, 188(4), 773-782.</ref><ref name="ref8">Gaj, T.; Gersbach, C.A.; Barbas, C.F. 3rd. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. ''Trends Biotechnol''., 2013, 31(7), 397-405.</ref>.

=== Applications: ===
==== Model organisms ====
ZFNs enable introduction of targeted modification in several model organisms used in common biological researches. Benefits of DSB repaired by cell’s endogenous mechanisms include: simple production of knock-out or knock-in cell lines, permanent and heritable mutation, compatibility with variety of species<ref name="ref8">Gaj, T.; Gersbach, C.A.; Barbas, C.F. 3rd. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. ''Trends Biotechnol''., 2013, 31(7), 397-405.</ref>.
==== Therapeutic application of ZFNs ====
ZFN-mediated gene modification by HDR has been used to correct mutations associated with X-linked severe combined immune deficiency (SCID), haemophilia B, sickle-cell disease and α1-antitrypsin deficiency. ZFN induced gene targeting by NHEJ repair has been shown as potential treatment for HIV/AIDS, either by targeting cellular co-receptors for HIV in primary T cells and hematopoietic stem/progenitor cells (clinical trials) or by targeting proviral HIV DNA<ref name="ref8">Gaj, T.; Gersbach, C.A.; Barbas, C.F. 3rd. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. ''Trends Biotechnol''., 2013, 31(7), 397-405.</ref>.

=== Potential problems ===
If the DNA-binding domain is not specific enough or target site is not unique in genome, ZFN may cleave at undesired location in the genome. Another potential problem is formation of ZFN homodimers that are by products of ZFN heterodimer that can cleave off-target. Besides cleaving off-target, ZFN homodimers mediate toxic effect. Undesired cleavage can lead to unwanted insertions or deletions at additional cleavage sites or vast number of DSBs can cause cell death. This can be avoided by extensive bioinformatics research beforehand, construction of ZFP that recognises longer and consequently rarer target sequences, using suitable linkers and cleavage domains that do not form unwanted homodimers<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref><ref name="ref2">Urnov, F. D.; Rebar, E.J.; Holmes, M. C.; Zhang, H. S.; Gregory, P.D. Genome editing with engineered zinc finger nucleases. ''Nat Rev Genet''., 2010, 11(9), 636-646.</ref>.

== AN IMPROVED ZINC-FINGER NUCLEASE ARCHITECTURE ==
Miller and colleagues present in their paper an improved ZFN architecture for more specific genome targeting. Their aim was to develop FokI cleavage domain variants that function as obligate heterodimers<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>.

=== Development of heterodimeric FokI cleavage domain variants ===
There are few things that need to be put in consideration when altering architecture of FokI DNA-cleavage domain. Firstly, FokI is an enzyme, this means goal is to improve interaction between domains but at the same time, catalytic function of domains must be conserved. Secondly, interaction between FokI DNA-cleavage domains in dimer has very low affinity, which is essential for ensuring cleavage specificity. Lastly, interface of FokI dimer is hydrophilic. Considering all the facts, Miller and colleagues developed strategy that involved step by step approach to modification of dimer interface coupled with direct testing of candidate variants for catalytic activity. Reporter system with green fluorescent protein (eGFP) was used to detect gene correction activity. The system uses HEK293 cell line containing a copy of the eGFP gene that is disrupted by short DNA fragment and has characterised ZFN target sequence [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F2.html (Figure 2a)]. Screening is based on ZFN cleavage of DNA fragment that is corrected by HDR using exogenous donor DNA containing missing eGFP sequence as template. If repair is successful, green fluorescence can be detected by fluorescence-activated cell sorting [http://commons.wikimedia.org/wiki/File:Fluorescence_Assisted_Cell_Sorting_(FACS)_B.jpg#mediaviewer/File:Fluorescence_Assisted_Cell_Sorting_(FACS)_B.jpg (FACS)]<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>. FACS is specialized type of flow cytometry, method for separating heterogeneous mixture of biological cells, which is based upon the specific light scattering at fluorescent wavelength<ref name="ref9">Flow cytometry; Wikipedia the free encylopedia [cited 2.1.2015]. http://en.wikipedia.org/wiki/Flow_cytometry#Fluorescence-activated_cell_sorting_.28FACS.29.</ref>. Results are shown in [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F2.html Figure 2b], left for cells transfected with donor DNA only and right for cells transfected with donor DNA and ZFNs. Step by step modification of interaction surface was carried out in four cycles. Modifications were implemented alternately, which means in each cycle only one partner domain was modified. Mutations were designed to form charge-charge interaction. Each variant was tested for gene correction activity as a heterodimer with unmodified partner ZFN and as a homodimer. More in detailed view of introduced mutation can be found in [http://www.nature.com/nbt/journal/v25/n7/suppinfo/nbt1319_S1.html Supplementary table 1]. In each cycle researchers identified a variant of domain that induced gene correction as a heterodimer and at the same time the domain had reduced activity as a homodimer [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F2.html (Figure 2d)]. Variants with best gene correction rate contained double mutations E490K:I538K and Q486E:I499L (also known as EL:KK). In one domain glutamic acid (E) at position 490 and isoleucine (I) at position 538 were replaced with lysine (K) (E490K:I538K), in partner domain glutamine (Q) at position 486 was replaced with glutamic acid and isoleucine at position 499 was replaced with leucine (L) (Q486E:I499L). As lysine is positively charged amino acid, variants with two additional lysine residues were referred to as ‘+’ and the other variants were referred to as ‘-‘, due to negative charge of introduced glutamic acid. Model of how variants may interact with each other is shown in [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F2.html Figure 2e]. Variants ‘+’ and ‘-‘ exhibited strong preference for heterodimerization and very weak preference for homodimerization when connected with suitable ZFP (L or R ZFP) in GFP reporter system. In [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F2.html Figure 2f] we can see that double mutations heterodimers (marked as L+/R- and L-/R+) have higher efficiency for gene correction than wild-type (wt) dimer (marked as Lwt/Rwt) and homodimers of double mutation variants (marked as L+/R+ and L-/R-) have almost none. Results of GFP reporter system indicate that formation of obligated heterodimer was achieved<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>.

=== Validation of heterodimeric FokI cleavage domain variants ===
==== Efficient endogenous gene editing ====
To confirm that the heterodimer variants preserved catalytic function researchers fused them with ZFPs that target a sequence present in exon 5 of the human gene for interleukin-2 receptor γ-chain (IL2Rγ). Designed ZFNs induced gene editing that introduced novel BsrBI restriction site in IL2Rγ [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F3.html (Figure 3a)]. Restriction enzyme BsrBI is able to cut at such restriction site resulting in additional band on electrophoresis. Results of gene editing efficiency shown in [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F3.html Figure 3b] indicate BsrBI restriction site was successfully inserted in case of ZFN with wt cleavage domain (Lwt/Rwt) and both heterodimeric combinations (L+/R- and L-/R+). Forced homodimers (L+/R+ and L-/R-) yielded no detectable gene editing. These results confirmed obligated heterodimerization of developed variants<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>.

==== Suppression of homodimer function ''in vitro'' ====
Cleavage activity ''in vitro'' was directly measured by radiolabeling of target sequences for ZFNs [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F4.html (Figure 4a)]. Target sequences were then digested by ZFNs with wt cleavage domain and variants of domain with double mutations, both in heterodimeric and homodimeric form. Migration of cleaved and uncleaved products in [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F4.html Figure 4b] indicate that wt cleavage domain (Lwt/Rwt) was active in heterodimeric and homomeric form, while efficient cleavage with double mutation variants (L+/R- and L-/R+) was only observed if domains formed heterodimer. This result once again supports development of variants, which form obligated heterodimer<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>.

==== Reduced levels of genome-wide cleavage ====
The aim of study was to develop more specific ZFNs that do not cleave off-target. Therefore, researchers conducted the next experiment on ZFN specificity of gene modification in mammalian cells. ZFN cleavage was determined by detection of proteins that localise at DNA DSBs. The proteins bound were detected by antibody-mediated technique. Protein that localise at DNA damage site and form foci, is tumour suppressor p53-binding protein 1 (53BP1). Protein 53BP1 was detected by immunofluorescence in target cells, which were transfected with ZFN expression constructs and treated with anti-53BP1 rabbit polyclonal antibodies. Results in [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F5.html Figure 5a] show that signal is weaker in cells transfected with ZFN with double mutations variants compared to ZFN with wt cleavage domain. This indicates cleavage activity was reduced in case of transfection with modified ZFN. Western blot analysis [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F5.html (Figure 5b)] confirmed that both ZFNs were expressed at comparable levels, suggesting that the reduced activity was a result of decreased off-target effect. Alternative damage marker is phosphorylated histone H2AX (γH2AX), which also forms foci at DSBs. Flow cytometry data [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F6.html (Figure 6a)] for target cells transfected with ZFNs and stained with antibodies against γH2AX showed that number of positive cells (cells forming γH2AX foci) was significantly lower for ZFNs with double mutations variants compared to ZFN with wt cleavage domain. In addition gene modification was monitored by Surveyor Nuclease assay, which determines the frequency of the small insertions and deletions (indels) characteristic of unspecific DSB repair by NHEJ. Results [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F6.html (Figure 6b)] showed comparable levels of gene modification in cells treated with ZFNs with wt and double mutations cleavage domains, indicating comparable cellular activities at target locus. These results confirmed that heterodimeric FokI cleavage domain variants retain catalytic activity while exhibiting reduced levels of off-target cleavage<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>.

=== Conclusion ===
In conclusion, Miller and colleagues developed two complementary FokI cleavage domain variants with double mutation, which function as an obligate heterodimer and improve ZFN specificity limiting off-target effect. Researchers predicted that their new architecture of FokI cleavage domain could be beneficial for therapeutic application of ZFNs due to additional mechanism for reducing off-target cleavage. It could also be helpful for less safety-intensive application such as crop engineering, cell-line engineering and construction of disease models. These ZFNs would be useful for gene modification protocols requiring simultaneous cleavage at multiple targets, where it would eliminate unwanted combination of ZFNs<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>.

== Further research ==
In 2011 same research group form Sangamo BioSciences published a [http://www.nature.com/nmeth/journal/v8/n1/full/nmeth.1539.html new paper] in Nature Methods discussing improved obligate heterodimeric architectures. In this study yeast-based selection system was used to cross-examine the dimer interface. A reporter assay was used to isolate FokI with mutations causing cold-sensitivity, phenotype resulting in diminished cleavage activity at lower temperatures but still be active at higher ones. The hypothesis of study was that cold-sensitive mutants would affect critical residues involved in dimerization, because this class of mutations is often associated with incorrect assembly of multisubunit protein complexes<ref name="ref10">Doyon, Y.; Vo, T.D.; Mendel, M.C.; Greenberg, S.G.; Wang, J.; Xia, D.F.; Miller, J.C.; Urnov, F.D.; Gregory, P.D.; Holmes, M.C. Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures. ''Nat Methods''., 2011, 8(1), 74-79.</ref>.

A selection system in ''Saccharomyces cerecisiae'' developed to isolate ZFN mutants with cold-sensitivity phenotype included two independent single-stranded annealing reporter constructs that were integrated into the yeast genome. DNA sequence for reporter genes MEL1 and PHO5 were interrupted by binding site for ZFN homodimer (CCR5-L) [http://www.nature.com/nmeth/journal/v8/n1/fig_tab/nmeth.1539_F1.html (Figure 1a)]. Additionally PHO5 reporter system had incorporated positive selection cassette natMX that enables resistance to antibiotic neuroseothricin. MEL1 reporter system contained the URA3 gene for negative selection using 5-fluoroorotic acid, which can be converted into the toxic compound causing cell death and kanMX cassette that enables resistance to antibiotic geneticin. ZFN-induced DSB would result in restoration of reporter gene expression and simultaneous elimination of all positive and negative selection markers. Library of FokI mutants was constructed by error-prone PCR that randomly mutagenized the sequence [http://www.nature.com/nmeth/journal/v8/n1/fig_tab/nmeth.1539_F1.html (Figure 1b)]. ZFN expression was induced at 22 °C, and then cells were collected and incubated in medium with geneticin and neurseothricin. With this step all cells carrying active ZFN constructs were eliminated. Remaining cells were shifted to 37 °C and plated on medium with 5-fluoroorotic acid and colorimetric substrate for Pho5. This step gave them conditionally active ZFNs. Constructs were then isolated and tested at 22 °C, 30 °C and 37 °C to confirm cold-sensitive phenotype [http://www.nature.com/nmeth/journal/v8/n1/fig_tab/nmeth.1539_F1.html (Figure 1c)]; 16 mutants had minimal activity at 22 °C but had restored reporter gene expression at higher temperature. Within isolated mutants previously altered residues that contributed to obligated heterodimerization (glutamine 486 and isoleucine 499 and 538) were found. Their focus was residues asparagine 496 and histidine 537, which face each other in dimerization interface. These two residues were substituted with a pair of oppositely charged amino acids into EL:KK FokI backbone. This substitution could strengthen dimerization in the context of the obligate heterodimer variants. The asparagine 496 was replaced with negatively charged glutamic and aspartic acid in the EL domain, and histidine 537 was replaced with positively charged lysine and arginine in the KK domain. Measured cleavage activity comparted to both wt and EL:KK versions suggested that aspartic acid 496 drove maximal activity in EL monomer, whereas lysine and arginine at position 537 resulted in similar activity improvements. This domains were referred to as ELD:KKK or ELD:KKR. Improved activity of new FokI variants was confirmed by various assays. All results supported superior cleavage activity and retainment of obligate heterodimer function. Researchers concluded that these enhanced FokI domains were portable to many ZFPs, independent of cell type and are a general solution for improved ZFN activity<ref name="ref10">Doyon, Y.; Vo, T.D.; Mendel, M.C.; Greenberg, S.G.; Wang, J.; Xia, D.F.; Miller, J.C.; Urnov, F.D.; Gregory, P.D.; Holmes, M.C. Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures. ''Nat Methods''., 2011, 8(1), 74-79.</ref>.

==References==
<references />

[[SB students resources]]

An improved zinc-finger nuclease architecture for highly specific genome editing

2015-01-11T16:22:16Z

EvaKnapič:

(Eva Knapič)

Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J.[http://www.nature.com/nbt/journal/v25/n7/full/nbt1319.html An improved zinc-finger nuclease architecture for highly specific genome editing]. ''Nat Biotechnol''., 2007, 25(7), 778-785.

== Genome editing ==
Genome editing is a type of genetic engineering in which DNA is inserted, removed or replaced from genome using artificially designed nucleases, enzymes that cleave the phosphodiester bonds between nucleotides. The principle of method is based on nuclease cleavage at a desired site of the genome in order to create specific double-stranded break, then put to use the cell’s endogenous mechanisms to repair the induced break by homology-directed repair or non-homologous end joining. There are a few different classes of engineered nucleases that are currently being used: zinc-finger nucleases, transcription activator-like effector nucleases and the CRISPR/Cas system. In this seminar we will focus on zinc finger nucleases. Firstly, we will take a look at basics of zinc-finger nucleases and their mechanism. Then we will discuss improved zinc-finger nuclease architecture for more specific efficiency described by Miller and colleagues in their paper published in Nature Biotechnology in 2007<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>.

== Zinc-finger nuclease ==
Zinc-finger nuclease (ZFN) is an artificially designed endonuclease that can be customised to cleave at sequence specific site on the DNA. The enzyme consists of two domains: DNA-binding domain composed of zinc finger structural motifs and DNA-cleavage domain of restriction enzyme FokI. Domains are connected with short inter-domain linker. This structure combines key qualities of DNA binding specificity, flexibility of zinc-finger motifs and cleavage activity of FokI catalytic domain that is robust but restricted. Furthermore all three elements can be optimised for retargeting and improving efficiency<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref><ref name="ref2">Urnov, F. D.; Rebar, E.J.; Holmes, M. C.; Zhang, H. S.; Gregory, P.D. Genome editing with engineered zinc finger nucleases. ''Nat Rev Genet''., 2010, 11(9), 636-646.</ref>.

=== DNA-binding domain ===
As mentioned above, zinc finger motifs represent DNA-binding domain of ZFN. [http://commons.wikimedia.org/wiki/File:Zinc_finger_rendered.png#mediaviewer/File:Zinc_finger_rendered.png Zinc fingers] are small protein structural motifs distinguished by coordinated zinc ions that stabilise the protein fold. Each zinc-finger recognises 3 base pairs (bp) of DNA. Contacts between the zinc fingers and DNA are made by α-helix that binds in the DNA major groove<ref name="ref3">Zinc finger ; Wikipedia the free encylopedia [cited 2.1.2015]. http://en.wikipedia.org/wiki/Zinc_finger.</ref>. Proteins that contain any number of zinc finger motifs are called zinc-finger proteins (ZFPs). Engineered ZFPs consist of three to six zinc finger motifs thus enabling binding of 9 bp to 18 bp targets. Longer recognition sequence improves specificity and precision of ZFPs. ZFP based DNA-binding domains can be coupled to various effector domains, which then cuts the DNA sequence determined by the ZFP<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref><ref name="ref2">Urnov, F. D.; Rebar, E.J.; Holmes, M. C.; Zhang, H. S.; Gregory, P.D. Genome editing with engineered zinc finger nucleases. ''Nat Rev Genet''., 2010, 11(9), 636-646.</ref>.

There are multiple approaches to designing new ZFPs with binding specificities chosen by user. The simplest and widely used method to generate new zinc finger sets is modular assembly. Candidate ZFPs for target sequence are obtained by determining individual zinc fingers that bind each triplet and assembling them together thus recognising the target sequence<ref name="ref2">Urnov, F. D.; Rebar, E.J.; Holmes, M. C.; Zhang, H. S.; Gregory, P.D. Genome editing with engineered zinc finger nucleases. ''Nat Rev Genet''., 2010, 11(9), 636-646.</ref>. Alternative methods for designing new ZFPs include two-finger modules, oligomerised pool engineering (OPEN) and Context-Dependent Assembly (CoDA). In two-finger modules instead of individual fingers, construct of two-fingers is used for recognising target DNA sequence. Advantage of this approach is better optimisation of finger junctions and speed of finding and assembling suitable zinc fingers, a limitation is extension of initial characterisation of two-finger units <ref name="ref4">Carroll, D. Genome engineering with zinc-finger nucleases. ''Genetics''., 2011, 188(4), 773-782.</ref>. The OPEN system approach is carried out in two steps, firstly appropriate finger pools are recombined to create small combinatorial library, then members of library that efficiently bind to the target site are isolated using bacterial two-hybrid selection method in which binding of a zinc finger domain to its target activates expression of selected marker gene<ref name="ref5">Maeder M. L. et al. Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification. ''Mol Cell''., 2008, 31(2), 294–301.</ref>. CoDA is publicly available platform of reagents and software. With this approach three-finger sets are assembled using N- and C- terminal fingers that have been previously identified in other arrays with common middle finger. CoDA does not treat fingers as independent modules but instead explicitly accounts for context-dependent effects between adjacent fingers, increasing the probability of efficiency <ref name="ref6">Sander J.D. et al. Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA). ''Nat Methods''., 2011, 8(1), 67-69.</ref>. Regardless of which approach is used for designing new ZFPs ''in vitro'', their application in living cells is not always successful. Reason is the complexity of genome that often contains naturally occurring multiple copies of sequence that are either identical or very similar to target sequence and these copies can act as additional targets for ZFNs. Another complication is chromatin structure at target sites that may obstruct cleavage<ref name="ref4">Carroll, D. Genome engineering with zinc-finger nucleases. ''Genetics''., 2011, 188(4), 773-782.</ref>.

=== Inter-domain linker ===
Inter-domain linker connects DNA-binding domain with DNA-cleavage domain. Length of the linker is responsible for the shaping of spacer. Spacer is short sequence between both recognition sites for DNA-binding domains, which enables precise definition of cleavage site. Wilson and colleagues have demonstrated that sites with 5 bp spacer lengths showed the most efficient ZFN mediated gene targeting when the linker is 2 or 4 amino acids and that sites with 3 or 4 bp spacers could not be targeted efficiently. Group also demonstrated that ZFN variants with 5 amino acids linker can efficiently target sites with a 7 bp spacer. However, the ideal linker with corresponding spacer can only be determined by taking into consideration both ZFN activity on the chromosomal target site and ZFN associated toxicity <ref name="ref7">Wilson, K.A.; McEwen, A.E.; Pruett-Miller, S.M.; Zhang, J.; Kildebeck, E.J.;, Porteus, M.H. Expanding the Repertoire of Target Sites for Zinc Finger Nuclease-mediated Genome Modification. Mol Ther Nucleic Acids., 2013, 2, e88.</ref>.

=== DNA-cleavage domain ===
DNA-cleavage domain of ZFN originates from FokI catalytic domain. FokI is type IIS restriction endonuclease consisting of an N-terminal DNA-binding domain and non-specific DNA-cleavage C-terminal domain. In order to cleave DNA two FokI catalytic domains must form dimer. Each subunit contains one catalytic centre that is responsible for cleaving one strand of DNA duplex. This characteristic of FokI catalytic domain is crucial for successful genome editing with ZFN. Dimerization requires two independent binding events, which must occur in correct orientation and spacing between monomers. DNA-cleavage domain is usually fused to the C-terminus of DNA-binding domain, thus two individual ZFNs must bind to opposite strands of DNA. This enables specific targeting of long and potentially unique recognition sites<ref name="ref2">Urnov, F. D.; Rebar, E.J.; Holmes, M. C.; Zhang, H. S.; Gregory, P.D. Genome editing with engineered zinc finger nucleases. ''Nat Rev Genet''., 2010, 11(9), 636-646.</ref>.

== ZFN-mediated genome editing ==
ZFNs create double-stranded breaks (DSBs) in DNA at sequence specific site. DSBs can be repaired by cell’s endogenous mechanisms: homology-directed repair (HDR) or non-homologous end joining (NHEJ). There are few different possible outcomes of DSB repair. If the break is repaired by NHEJ, it can lead to gene disruption, tag ligation or large deletion. Gene disruption occurs when two broken ends are ligated back together that can lead to small insertions or deletions at the break site causing target gene disruption. Tag ligation occurs when adaptor with sticky ends is provided and is ligated into the chromosome producing tagged allele. When two different ZFNs cleave simultaneous same chromosome, entire segment between them is deleted. If the break is repaired by HDR, which occurs in the presence of donor DNA, it can result in gene correction, targeted gene addition or transgene stacking. If donor with small change, for example single bp, is provided, editing of endogenous allele results in gene correction. This enables easier functional genomics studies, modelling of disease caused by point mutation and gene therapy research. If provided donor carries transgene and has specific homology arms, transgene can be efficiently integrated into chromosome at break site, which results in targeted gene addition. Transgene stacking occurs when provided donor carries multiple linked transgenes between the homology arms. With this approach not only gene addition can be done, but also gene correction if DSB is created within or near gene in question and provided donor carries modified gene sequence<ref name="ref2">Urnov, F. D.; Rebar, E.J.; Holmes, M. C.; Zhang, H. S.; Gregory, P.D. Genome editing with engineered zinc finger nucleases. ''Nat Rev Genet''., 2010, 11(9), 636-646.</ref><ref name="ref4">Carroll, D. Genome engineering with zinc-finger nucleases. ''Genetics''., 2011, 188(4), 773-782.</ref><ref name="ref8">Gaj, T.; Gersbach, C.A.; Barbas, C.F. 3rd. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. ''Trends Biotechnol''., 2013, 31(7), 397-405.</ref>.

=== Applications: ===
==== Model organisms ====
ZFNs enable introduction of targeted modification in several model organisms used in common biological researches. Benefits of DSB repaired by cell’s endogenous mechanisms include: simple production of knock-out or knock-in cell lines, permanent and heritable mutation, compatibility with variety of species<ref name="ref8">Gaj, T.; Gersbach, C.A.; Barbas, C.F. 3rd. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. ''Trends Biotechnol''., 2013, 31(7), 397-405.</ref>.
==== Therapeutic application of ZFNs ====
ZFN-mediated gene modification by HDR has been used to correct mutations associated with X-linked severe combined immune deficiency (SCID), haemophilia B, sickle-cell disease and α1-antitrypsin deficiency. ZFN induced gene targeting by NHEJ repair has been shown as potential treatment for HIV/AIDS, either by targeting cellular co-receptors for HIV in primary T cells and hematopoietic stem/progenitor cells (clinical trials) or by targeting proviral HIV DNA<ref name="ref8">Gaj, T.; Gersbach, C.A.; Barbas, C.F. 3rd. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. ''Trends Biotechnol''., 2013, 31(7), 397-405.</ref>.

=== Potential problems ===
If the DNA-binding domain is not specific enough or target site is not unique in genome, ZFN may cleave at undesired location in the genome. Another potential problem is formation of ZFN homodimers that are by products of ZFN heterodimer that can cleave off-target. Besides cleaving off-target, ZFN homodimers mediate toxic effect. Undesired cleavage can lead to unwanted insertions or deletions at additional cleavage sites or vast number of DSBs can cause cell death. This can be avoided by extensive bioinformatics research beforehand, construction of ZFP that recognises longer and consequently rarer target sequences, using suitable linkers and cleavage domains that do not form unwanted homodimers<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref><ref name="ref2">Urnov, F. D.; Rebar, E.J.; Holmes, M. C.; Zhang, H. S.; Gregory, P.D. Genome editing with engineered zinc finger nucleases. ''Nat Rev Genet''., 2010, 11(9), 636-646.</ref>.

== AN IMPROVED ZINC-FINGER NUCLEASE ARCHITECTURE ==
Miller and colleagues present in their paper an improved ZFN architecture for more specific genome targeting. Their aim was to develop FokI cleavage domain variants that function as obligate heterodimers<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>.

=== Development of heterodimeric FokI cleavage domain variants ===
There are few things that need to be put in consideration when altering architecture of FokI DNA-cleavage domain. Firstly, FokI is an enzyme, this means goal is to improve interaction between domains but at the same time, catalytic function of domains must be conserved. Secondly, interaction between FokI DNA-cleavage domains in dimer has very low affinity, which is essential for ensuring cleavage specificity. Lastly, interface of FokI dimer is hydrophilic. Considering all the facts, Miller and colleagues developed strategy that involved step by step approach to modification of dimer interface coupled with direct testing of candidate variants for catalytic activity. Reporter system with green fluorescent protein (eGFP) was used to detect gene correction activity. The system uses HEK293 cell line containing a copy of the eGFP gene that is disrupted by short DNA fragment and has characterised ZFN target sequence [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F2.html (Figure 2a)]. Screening is based on ZFN cleavage of DNA fragment that is corrected by HDR using exogenous donor DNA containing missing eGFP sequence as template. If repair is successful, green fluorescence can be detected by fluorescence-activated cell sorting [http://commons.wikimedia.org/wiki/File:Fluorescence_Assisted_Cell_Sorting_(FACS)_B.jpg#mediaviewer/File:Fluorescence_Assisted_Cell_Sorting_(FACS)_B.jpg (FACS)]<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>. FACS is specialized type of flow cytometry, method for separating heterogeneous mixture of biological cells, which is based upon the specific light scattering at fluorescent wavelength<ref name="ref9">Flow cytometry; Wikipedia the free encylopedia [cited 2.1.2015]. http://en.wikipedia.org/wiki/Flow_cytometry#Fluorescence-activated_cell_sorting_.28FACS.29.</ref>. Results are shown in [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F2.html Figure 2b], left for cells transfected with donor DNA only and right for cells transfected with donor DNA and ZFNs. Step by step modification of interaction surface was carried out in four cycles. Modifications were implemented alternately, which means in each cycle only one partner domain was modified. Mutations were designed to form charge-charge interaction. Each variant was tested for gene correction activity as a heterodimer with unmodified partner ZFN and as a homodimer. More in detailed view of introduced mutation can be found in [http://www.nature.com/nbt/journal/v25/n7/suppinfo/nbt1319_S1.html Supplementary table 1]. In each cycle researchers identified a variant of domain that induced gene correction as a heterodimer and at the same time the domain had reduced activity as a homodimer [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F2.html (Figure 2d)]. Variants with best gene correction rate contained double mutations E490K:I538K and Q486E:I499L (also known as EL:KK). In one domain glutamic acid (E) at position 490 and isoleucine (I) at position 538 were replaced with lysine (K) (E490K:I538K), in partner domain glutamine (Q) at position 486 was replaced with glutamic acid and isoleucine at position 499 was replaced with leucine (L) (Q486E:I499L). As lysine is positively charged amino acid, variants with two additional lysine residues were referred to as ‘+’ and the other variants were referred to as ‘-‘, due to negative charge of introduced glutamic acid. Model of how variants may interact with each other is shown in [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F2.html Figure 2e]. Variants ‘+’ and ‘-‘ exhibited strong preference for heterodimerization and very weak preference for homodimerization when connected with suitable ZFP (L or R ZFP) in GFP reporter system. In [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F2.html Figure 2f] we can see that double mutations heterodimers (marked as L+/R- and L-/R+) have higher efficiency for gene correction than wild-type (wt) dimer (marked as Lwt/Rwt) and homodimers of double mutation variants (marked as L+/R+ and L-/R-) have almost none. Results of GFP reporter system indicate that formation of obligated heterodimer was achieved<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>.

=== Validation of heterodimeric FokI cleavage domain variants ===
==== Efficient endogenous gene editing ====
To confirm that the heterodimer variants preserved catalytic function researchers fused them with ZFPs that target a sequence present in exon 5 of the human gene for interleukin-2 receptor γ-chain (IL2Rγ). Designed ZFNs induced gene editing that introduced novel BsrBI restriction site in IL2Rγ [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F3.html (Figure 3a)]. Restriction enzyme BsrBI is able to cut at such restriction site resulting in additional band on electrophoresis. Results of gene editing efficiency shown in [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F3.html Figure 3b] indicate BsrBI restriction site was successfully inserted in case of ZFN with wt cleavage domain (Lwt/Rwt) and both heterodimeric combinations (L+/R- and L-/R+). Forced homodimers (L+/R+ and L-/R-) yielded no detectable gene editing. These results confirmed obligated heterodimerization of developed variants<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>.

==== Suppression of homodimer function ''in vitro'' ====
Cleavage activity ''in vitro'' was directly measured by radiolabeling of target sequences for ZFNs [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F4.html (Figure 4a)]. Target sequences were then digested by ZFNs with wt cleavage domain and variants of domain with double mutations, both in heterodimeric and homodimeric form. Migration of cleaved and uncleaved products in [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F4.html Figure 4b] indicate that wt cleavage domain (Lwt/Rwt) was active in heterodimeric and homomeric form, while efficient cleavage with double mutation variants (L+/R- and L-/R+) was only observed if domains formed heterodimer. This result once again supports development of variants, which form obligated heterodimer<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>.

==== Reduced levels of genome-wide cleavage ====
The aim of study was to develop more specific ZFNs that do not cleave off-target. Therefore, researchers conducted the next experiment on ZFN specificity of gene modification in mammalian cells. ZFN cleavage was determined by detection of proteins that localise at DNA DSBs. The proteins bound were detected by antibody-mediated technique. Protein that localise at DNA damage site and form foci, is tumour suppressor p63-binding protein 1 (53BP1). Protein 53BP1 was detected by immunofluorescence in target cells, which were transfected with ZFN expression constructs and treated with anti-53BP1 rabbit polyclonal antibodies. Results in [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F5.html Figure 5a] show that signal is weaker in cells transfected with ZFN with double mutations variants compared to ZFN with wt cleavage domain. This indicates cleavage activity was reduced in case of transfection with modified ZFN. Western blot analysis [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F5.html (Figure 5b)] confirmed that both ZFNs were expressed at comparable levels, suggesting that the reduced activity was a result of decreased off-target effect. Alternative damage marker is phosphorylated histone H2AX (γH2AX), which also forms foci at DSBs. Flow cytometry data [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F6.html (Figure 6a)] for target cells transfected with ZFNs and stained with antibodies against γH2AX showed that number of positive cells (cells forming γH2AX foci) was significantly lower for ZFNs with double mutations variants compared to ZFN with wt cleavage domain. In addition gene modification was monitored by Surveyor Nuclease assay, which determines the frequency of the small insertions and deletions (indels) characteristic of unspecific DSB repair by NHEJ. Results [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F6.html (Figure 6b)] showed comparable levels of gene modification in cells treated with ZFNs with wt and double mutations cleavage domains, indicating comparable cellular activities at target locus. These results confirmed that heterodimeric FokI cleavage domain variants retain catalytic activity while exhibiting reduced levels of off-target cleavage<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>.

=== Conclusion ===
In conclusion, Miller and colleagues developed two complementary FokI cleavage domain variants with double mutation, which function as an obligate heterodimer and improve ZFN specificity limiting off-target effect. Researchers predicted that their new architecture of FokI cleavage domain could be beneficial for therapeutic application of ZFNs due to additional mechanism for reducing off-target cleavage. It could also be helpful for less safety-intensive application such as crop engineering, cell-line engineering and construction of disease models. These ZFNs would be useful for gene modification protocols requiring simultaneous cleavage at multiple targets, where it would eliminate unwanted combination of ZFNs<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>.

== Further research ==
In 2011 same research group form Sangamo BioSciences published a [http://www.nature.com/nmeth/journal/v8/n1/full/nmeth.1539.html new paper] in Nature Methods discussing improved obligate heterodimeric architectures. In this study yeast-based selection system was used to cross-examine the dimer interface. A reporter assay was used to isolate FokI with mutations causing cold-sensitivity, phenotype resulting in diminished cleavage activity at lower temperatures but still be active at higher ones. The hypothesis of study was that cold-sensitive mutants would affect critical residues involved in dimerization, because this class of mutations is often associated with incorrect assembly of multisubunit protein complexes<ref name="ref10">Doyon, Y.; Vo, T.D.; Mendel, M.C.; Greenberg, S.G.; Wang, J.; Xia, D.F.; Miller, J.C.; Urnov, F.D.; Gregory, P.D.; Holmes, M.C. Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures. ''Nat Methods''., 2011, 8(1), 74-79.</ref>.

A selection system in ''Saccharomyces cerecisiae'' developed to isolate ZFN mutants with cold-sensitivity phenotype included two independent single-stranded annealing reporter constructs that were integrated into the yeast genome. DNA sequence for reporter genes MEL1 and PHO5 were interrupted by binding site for ZFN homodimer (CCR5-L) [http://www.nature.com/nmeth/journal/v8/n1/fig_tab/nmeth.1539_F1.html (Figure 1a)]. Additionally PHO5 reporter system had incorporated positive selection cassette natMX that enables resistance to antibiotic neuroseothricin. MEL1 reporter system contained the URA3 gene for negative selection using 5-fluoroorotic acid, which can be converted into the toxic compound causing cell death and kanMX cassette that enables resistance to antibiotic geneticin. ZFN-induced DSB would result in restoration of reporter gene expression and simultaneous elimination of all positive and negative selection markers. Library of FokI mutants was constructed by error-prone PCR that randomly mutagenized the sequence [http://www.nature.com/nmeth/journal/v8/n1/fig_tab/nmeth.1539_F1.html (Figure 1b)]. ZFN expression was induced at 22 °C, and then cells were collected and incubated in medium with geneticin and neurseothricin. With this step all cells carrying active ZFN constructs were eliminated. Remaining cells were shifted to 37 °C and plated on medium with 5-fluoroorotic acid and colorimetric substrate for Pho5. This step gave them conditionally active ZFNs. Constructs were then isolated and tested at 22 °C, 30 °C and 37 °C to confirm cold-sensitive phenotype [http://www.nature.com/nmeth/journal/v8/n1/fig_tab/nmeth.1539_F1.html (Figure 1c)]; 16 mutants had minimal activity at 22 °C but had restored reporter gene expression at higher temperature. Within isolated mutants previously altered residues that contributed to obligated heterodimerization (glutamine 486 and isoleucine 499 and 538) were found. Their focus was residues asparagine 496 and histidine 537, which face each other in dimerization interface. These two residues were substituted with a pair of oppositely charged amino acids into EL:KK FokI backbone. This substitution could strengthen dimerization in the context of the obligate heterodimer variants. The asparagine 496 was replaced with negatively charged glutamic and aspartic acid in the EL domain, and histidine 537 was replaced with positively charged lysine and arginine in the KK domain. Measured cleavage activity comparted to both wt and EL:KK versions suggested that aspartic acid 496 drove maximal activity in EL monomer, whereas lysine and arginine at position 537 resulted in similar activity improvements. This domains were referred to as ELD:KKK or ELD:KKR. Improved activity of new FokI variants was confirmed by various assays. All results supported superior cleavage activity and retainment of obligate heterodimer function. Researchers concluded that these enhanced FokI domains were portable to many ZFPs, independent of cell type and are a general solution for improved ZFN activity<ref name="ref10">Doyon, Y.; Vo, T.D.; Mendel, M.C.; Greenberg, S.G.; Wang, J.; Xia, D.F.; Miller, J.C.; Urnov, F.D.; Gregory, P.D.; Holmes, M.C. Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures. ''Nat Methods''., 2011, 8(1), 74-79.</ref>.

==References==
<references />

[[SB students resources]]

SB students resources

2015-01-11T12:50:54Z

EvaKnapič:

===Introduction to our students resources in Synthetic Biology===
(Marko Dolinar)

Synthetic biology made a vast progress in good 10 years since it established itself as an interdisciplinary field of research on the interface of molecular biology and engineering. University of Ljubljana Faculty of Chemistry and Chemical Technology has introduced a Synthetic Biology course as a part od Biochemistry MSc programme only in 2013/14. This is relatively late, considering a great success of Slovenian students at iGEM competitions since their first attendance in 2006. On the other hand, the field is still in its first stages if development and a complete textbook for a MSc level course is still missing. This is the reason why our students collaborated on the preparation of a Synthetic Biology textbook with the working title Synthetic Biology - A Students Textbook. It exists as a draft that is not publicly available and is actually part 1 of a (to be) 2-volumes title. Part I is subtitled Engineering Biology, while Part II (that currently doesn't exisist yet) will be subtitled Synthetic Biology Applications.

As in all highly competitive fields of science and technology, students should be following recent progress by reading articles in high quality journals. However, this is often a very difficult task, especially at the BSc level. Specificities of the scientific and technical language, push of publishers towards very short methodological chapters and limited knowledge studens might have about advanced techniques make understanding papers a very challenging task. Therefore, I decided to face MSc students with the challenge to explain selected SB articles in a manner that would make the content of these articles understandable to BSc level students and non-experts.

In 2014/15, seminars in Synthetic Biology include explanations and presentations of some of the top-cited articles from the field of Synthetic Biology. I compiled a list of 95 articles published between 2000 and 2014 having the highest number of citations according to the Web of Science database. The list ended with the paper just exceeding the 100 citations limit. Not included in the list were reviews. With 20 students enrolled in the course, the list has been further reduced to top 40 papers in the field. Students have been asked to check for content (they further eliminated 3 papers which proved to be reviews) and availabitly (they all seemed to be available as full texts with our university subscriptions). My suggestion was to avoid selecting for presentation papers with very similar content. Especially in the field of genome editing there has been a very rapid progress in the past few years resulting in a number of highly-cited articles which could appear very similar in content for a non-specialist. From the shortlist of 37 articles, students selected a topic they believed would be most interesting or easiest to explain. Presentations will be both written (in English, which is not the mother tongue of my students) and oral (in Slovenian, to establish and maintain Slovenian terminology in the field).

===List of articles for presentation===

This is the list of top-cited papers from the broader field of Synthetic Biology that students chose for explanation in 2014/15 (sorted by year of publication):

#[[A synthetic oscillatory network of transcriptional regulators]], Michael B. Elowitz & Stanislas Leibler, Letters to Nature, 2000 - Valter Bergant
#[[Construction of a genetic toggle switch in Escherichia coli]]. Gardner ''et al''., Nature, 2000 - Urban Bezeljak
#Positive feedback in eukaryotic gene networks: cell differentiation by graded to binary response conversion (2001) - Andreja Bratovš
#Chemical synthesis of poliovirus cDNA: Generation of infectious virus in the absence of natural template (2002) - Veronika Jarc
#[[Combinatorial synthesis of genetic networks]]. Guet C.C. ''et al'', Science, 2002 - Maja Remškar
#Engineering a mevalonate pathway in Escherichia coli for production of terpenoids (2003) - Ana Kapraljević
#Programmed population control by cell-cell communication and regulated killing (2004) - Alja Zottel
#Gene regulation at the single-cell level (2005) - Katarina Uršič
#[[A synthetic multicellular system for programmed pattern formation]]. (2005) - Mitja Crček
#Long-term monitoring of bacteria undergoing programmed population control in a microchemostat (2005) - Jana Verbančič
#Tuning genetic control through promoter engineering (2005) - Špela Pohleven
#Production of the antimalarial drug precursor artemisinic acid in engineered yeast (2006) - Živa Marsetič
#[[An improved zinc-finger nuclease architecture for highly specific genome editing]], Miller ''et al''., ''Nature Biotechnol''., 2007 - Eva Knapič
#Establishment of HIV-1 resistance in CD4(+) T cells by genome editing using zinc-finger nucleases (2008) - Tamara Marić
#Synthetic protein scaffolds provide modular control over metabolic flux (2009) - Ana Dolinar
#[[Creation of a bacterial cell controlled by a chemically synthesized genome]]. Gibson, D. G. ''et al.'', Science, 2010 - Eva Lucija Kozak
#A TALE nuclease architecture for efficient genome editing (2011) Jernej Mustar
#Multiplex genome engineering using CRISPR/Cas systems (2013) - Uroš Stupar
#[[RNA-guided human genome engineering via Cas9]]. Mali ''et al''., Science, 2013 - Luka Smole
#[[One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering (2013)]] - Andrej Vrankar

''Please link the title of each paper with your written seminar wiki page. Expand the citation according to the following example:
''
#Emergent bistability by a growth-modulating positive feedback circuit. Tan et al., Nature Chem. Biol., 2009

An improved zinc-finger nuclease architecture for highly specific genome editing

2015-01-11T12:42:23Z

EvaKnapič:

(Eva Knapič)

Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J.[http://www.nature.com/nbt/journal/v25/n7/full/nbt1319.html An improved zinc-finger nuclease architecture for highly specific genome editing]. ''Nat Biotechnol''., 2007, 25(7), 778-785.

== Genome editing ==
Genome editing is a type of genetic engineering in which DNA is inserted, removed or replaced from genome using artificially designed nucleases, enzymes that cleave the phosphodiester bonds between nucleotides. The principle of method is based on nuclease cleavage at a desired site of the genome in order to create specific double-stranded break, then put to use the cell’s endogenous mechanisms to repair the induced break by homology-directed repair or non-homologous end joining. There are a few different classes of engineered nucleases that are currently being used: zinc-finger nucleases, transcription activator-like effector nucleases and the CRISPR/Cas system. In this seminar we will focus on zinc finger nucleases. Firstly, we will take a look at basics of zinc-finger nucleases and their mechanism. Then we will discuss improved zinc-finger nuclease architecture for more specific efficiency described by Miller and colleagues in their paper published in Nature Biotechnology in 2007<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>.

== Zinc-finger nuclease ==
Zinc-finger nuclease (ZFN) is an artificially designed endonuclease that can be customised to cleave at sequence specific site on the DNA. The enzyme consists of two domains: DNA-binding domain composed of zinc finger structural motifs and DNA-cleavage domain of restriction enzyme FokI. Domains are connected with short inter-domain linker. This structure combines key qualities of DNA binding specificity, flexibility of zinc-finger motifs and cleavage activity of FokI catalytic domain that is robust but restricted. Furthermore all three elements can be optimised for retargeting and improving efficiency<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref><ref name="ref2">Urnov, F. D.; Rebar, E.J.; Holmes, M. C.; Zhang, H. S.; Gregory, P.D. Genome editing with engineered zinc finger nucleases. ''Nat Rev Genet''., 2010, 11(9), 636-646.</ref>.

=== DNA-binding domain ===
As mentioned above, zinc finger motifs represent DNA-binding domain of ZFN. [http://commons.wikimedia.org/wiki/File:Zinc_finger_rendered.png#mediaviewer/File:Zinc_finger_rendered.png Zinc fingers] are small protein structural motifs distinguished by coordinated zinc ions that stabilise the protein fold. Each zinc-finger recognises 3 base pairs (bp) of DNA. Contacts between the zinc fingers and DNA are made by α-helix that binds in the DNA major groove<ref name="ref3">Zinc finger ; Wikipedia the free encylopedia [cited 2.1.2015]. http://en.wikipedia.org/wiki/Zinc_finger.</ref>. Proteins that contain any number of zinc finger motifs are called zinc-finger proteins (ZFPs). Engineered ZFPs consist of three to six zinc finger motifs thus enabling binding of 9 bp to 18 bp targets. Longer recognition sequence improves specificity and precision of ZFPs. ZFP based DNA-binding domains can be coupled to various effector domains, which then cuts the DNA sequence determined by the ZFP<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref><ref name="ref2">Urnov, F. D.; Rebar, E.J.; Holmes, M. C.; Zhang, H. S.; Gregory, P.D. Genome editing with engineered zinc finger nucleases. ''Nat Rev Genet''., 2010, 11(9), 636-646.</ref>.

There are multiple approaches to designing new ZFPs with binding specificities chosen by user. The simplest and widely used method to generate new zinc finger sets is modular assembly. Candidate ZFPs for target sequence are obtained by determining individual zinc fingers that bind each triplet and assembling them together thus recognising the target sequence<ref name="ref2">Urnov, F. D.; Rebar, E.J.; Holmes, M. C.; Zhang, H. S.; Gregory, P.D. Genome editing with engineered zinc finger nucleases. ''Nat Rev Genet''., 2010, 11(9), 636-646.</ref>. Alternative methods for designing new ZFPs include two-finger modules, oligomerised pool engineering (OPEN) and Context-Dependent Assembly (CoDA). In two-finger modules instead of individual fingers, construct of two-fingers is used for recognising target DNA sequence. Advantage of this approach is better optimisation of finger junctions and speed of finding and assembling suitable zinc fingers, a limitation is extension of initial characterisation of two-finger units <ref name="ref4">Carroll, D. Genome engineering with zinc-finger nucleases. ''Genetics''., 2011, 188(4), 773-782.</ref>. The OPEN system approach is carried out in two steps, firstly appropriate finger pools are recombined to create small combinatorial library, then members of library that efficiently bind to the target site are isolated using bacterial two-hybrid selection method in which binding of a zinc finger domain to its target activates expression of selected marker gene<ref name="ref5">Maeder M. L. et al. Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification. ''Mol Cell''., 2008, 31(2), 294–301.</ref>. CoDA is publicly available platform of reagents and software. With this approach three-finger sets are assembled using N- and C- terminal fingers that have been previously identified in other arrays with common middle finger. CoDA does not treat fingers as independent modules but instead explicitly accounts for context-dependent effects between adjacent fingers, increasing the probability of efficiency <ref name="ref6">Sander J.D. et al. Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA). ''Nat Methods''., 2011, 8(1), 67-69.</ref>. Regardless of which approach is used for designing new ZFPs ''in vitro'', their application in living cells is not always successful. Reason is the complexity of genome that often contains naturally occurring multiple copies of sequence that are either identical or very similar to target sequence and these copies can act as additional targets for ZFNs. Another complication is chromatin structure at target sites that may obstruct cleavage<ref name="ref4">Carroll, D. Genome engineering with zinc-finger nucleases. ''Genetics''., 2011, 188(4), 773-782.</ref>.

=== Inter-domain linker ===
Inter-domain linker connects DNA-binding domain with DNA-cleavage domain. Length of the linker is responsible for the shaping of spacer. Spacer is short sequence between both recognition sites for DNA-binding domains, which enables precise definition of cleavage site. Wilson and colleagues have demonstrated that sites with 5 bp spacer lengths showed the most efficient ZFN mediated gene targeting when the linker is 2 or 4 amino acids and that sites with 3 or 4 bp spacers could not be targeted efficiently. Group also demonstrated that ZFN variants with 5 amino acids linker can efficiently target sites with a 7 bp spacer. However, the ideal linker with corresponding spacer can only be determined by taking into consideration both ZFN activity on the chromosomal target site and ZFN associated toxicity <ref name="ref7">Wilson, K.A.; McEwen, A.E.; Pruett-Miller, S.M.; Zhang, J.; Kildebeck, E.J.;, Porteus, M.H. Expanding the Repertoire of Target Sites for Zinc Finger Nuclease-mediated Genome Modification. Mol Ther Nucleic Acids., 2013, 2, e88.</ref>.

=== DNA-cleavage domain ===
DNA-cleavage domain of ZFN originates from FokI catalytic domain. FokI is type IIS restriction endonuclease consisting of an N-terminal DNA-binding domain and non-specific DNA-cleavage C-terminal domain. In order to cleave DNA two FokI catalytic domains must form dimer. Each subunit contains one catalytic centre that is responsible for cleaving one strand of DNA duplex. This characteristic of FokI catalytic domain is crucial for successful genome editing with ZFN. Dimerization requires two independent binding events, which must occur in correct orientation and spacing between monomers. DNA-cleavage domain is usually fused to the C-terminus of DNA-binding domain, thus two individual ZFNs must bind to opposite strands of DNA. This enables specific targeting of long and potentially unique recognition sites<ref name="ref2">Urnov, F. D.; Rebar, E.J.; Holmes, M. C.; Zhang, H. S.; Gregory, P.D. Genome editing with engineered zinc finger nucleases. ''Nat Rev Genet''., 2010, 11(9), 636-646.</ref>.

== ZFN-mediated genome editing ==
ZFNs create double-stranded breaks (DSBs) in DNA at sequence specific site. DSBs can be repaired by cell’s endogenous mechanisms: homology-directed repair (HDR) or non-homologous end joining (NHEJ). There are few different possible outcomes of DSB repair. If the break is repaired by NHEJ, it can lead to gene disruption, tag ligation or large deletion. Gene disruption occurs when two broken ends are ligated back together that can lead to small insertions or deletions at the break site causing target gene disruption. Tag ligation occurs when adaptor with sticky ends is provided and is ligated into the chromosome producing tagged allele. When two different ZFNs cleave simultaneous same chromosome, entire segment between them is deleted. If the break is repaired by HDR, which occurs in the presence of donor DNA, it can result in gene correction, targeted gene addition or transgene stacking. If donor with small change, for example single bp, is provided, editing of endogenous allele results in gene correction. This enables easier functional genomics studies, modelling of disease caused by point mutation and gene therapy research. If provided donor carries transgene and has specific homology arms, transgene can be efficiently integrated into chromosome at break site, which results in targeted gene addition. Transgene stacking occurs when provided donor carries multiple linked transgenes between the homology arms. With this approach not only gene addition can be done, but also gene correction if DSB is created within or near gene in question and provided donor carries modified gene sequence<ref name="ref2">Urnov, F. D.; Rebar, E.J.; Holmes, M. C.; Zhang, H. S.; Gregory, P.D. Genome editing with engineered zinc finger nucleases. ''Nat Rev Genet''., 2010, 11(9), 636-646.</ref><ref name="ref4">Carroll, D. Genome engineering with zinc-finger nucleases. ''Genetics''., 2011, 188(4), 773-782.</ref><ref name="ref8">Gaj, T.; Gersbach, C.A.; Barbas, C.F. 3rd. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. ''Trends Biotechnol''., 2013, 31(7), 397-405.</ref>.

=== Applications: ===
==== Model organisms ====
ZFNs enable introduction of targeted modification in several model organisms used in common biological researches. Benefits of DSB repaired by cell’s endogenous mechanisms include: simple production of knock-out or knock-in cell lines, permanent and heritable mutation, compatibility with variety of species<ref name="ref8">Gaj, T.; Gersbach, C.A.; Barbas, C.F. 3rd. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. ''Trends Biotechnol''., 2013, 31(7), 397-405.</ref>.
==== Therapeutic application of ZFNs ====
ZFN-mediated gene modification by HDR has been used to correct mutations associated with X-linked severe combined immune deficiency (SCID), haemophilia B, sickle-cell disease and α1-antitrypsin deficiency. ZFN induced gene targeting by NHEJ repair has been shown as potential treatment for HIV/AIDS, either by targeting cellular co-receptors for HIV in primary T cells and hematopoietic stem/progenitor cells (clinical trials) or by targeting proviral HIV DNA<ref name="ref8">Gaj, T.; Gersbach, C.A.; Barbas, C.F. 3rd. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. ''Trends Biotechnol''., 2013, 31(7), 397-405.</ref>.

=== Potential problems ===
If the DNA-binding domain is not specific enough or target site is not unique in genome, ZFN may cleave at undesired location in the genome. Another potential problem is formation of ZFN homodimers that are by products of ZFN heterodimer that can cleave off-target. Besides cleaving off-target, ZFN homodimers mediate toxic effect. Undesired cleavage can lead to unwanted insertions or deletions at additional cleavage sites or vast number of DSBs can cause cell death. This can be avoided by extensive bioinformatics research beforehand, construction of ZFP that recognises longer and consequently rarer target sequences, usingf suitable linkers and cleavage domains that do not form unwanted homodimers<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref><ref name="ref2">Urnov, F. D.; Rebar, E.J.; Holmes, M. C.; Zhang, H. S.; Gregory, P.D. Genome editing with engineered zinc finger nucleases. ''Nat Rev Genet''., 2010, 11(9), 636-646.</ref>.

== AN IMPROVED ZINC-FINGER NUCLEASE ARCHITECTURE ==
Miller and colleagues present in their paper an improved ZFN architecture for more specific genome targeting. Their aim was to develop FokI cleavage domain variants that function as obligate heterodimers<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>.

=== Development of heterodimeric FokI cleavage domain variants ===
There are few things that need to be put in consideration when altering architecture of FokI DNA-cleavage domain. Firstly, FokI is an enzyme, this means goal is to improve interaction between domains but at the same time, catalytic function of domains must be conserved. Secondly, interaction between FokI DNA-cleavage domains in dimer has very low affinity, which is essential for ensuring cleavage specificity. Lastly, interface of FokI dimer is hydrophilic. Considering all the facts, Miller and colleagues developed strategy that involved step by step approach to modification of dimer interface coupled with direct testing of candidate variants for catalytic activity. Reporter system with green fluorescent protein (eGFP) was used to detect gene correction activity. The system uses HEK293 cell line containing a copy of the eGFP gene that is disrupted by short DNA fragment and has characterised ZFN target sequence [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F2.html (Figure 2a)]. Screening is based on ZFN cleavage of DNA fragment that is corrected by HDR using exogenous donor DNA containing missing eGFP sequence as template. If repair is successful, green fluorescence can be detected by fluorescence-activated cell sorting [http://commons.wikimedia.org/wiki/File:Fluorescence_Assisted_Cell_Sorting_(FACS)_B.jpg#mediaviewer/File:Fluorescence_Assisted_Cell_Sorting_(FACS)_B.jpg (FACS)]<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>. FACS is specialized type of flow cytometry, method for separating heterogeneous mixture of biological cells, which is based upon the specific light scattering at fluorescent wavelength<ref name="ref9">Flow cytometry; Wikipedia the free encylopedia [cited 2.1.2015]. http://en.wikipedia.org/wiki/Flow_cytometry#Fluorescence-activated_cell_sorting_.28FACS.29.</ref>. Results are shown in [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F2.html Figure 2b], left for cells transfected with donor DNA only and right for cells transfected with donor DNA and ZFNs. Step by step modification of interaction surface was carried out in four cycles. Modifications were implemented alternately, which means in each cycle only one partner domain was modified. Mutations were designed to form charge-charge interaction. Each variant was tested for gene correction activity as a heterodimer with unmodified partner ZFN and as a homodimer. More in detailed view of introduced mutation can be found in [http://www.nature.com/nbt/journal/v25/n7/suppinfo/nbt1319_S1.html Supplementary table 1]. In each cycle researchers identified a variant of domain that induced gene correction as a heterodimer and at the same time the domain had reduced activity as a homodimer [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F2.html (Figure 2d)]. Variants with best gene correction rate contained double mutations E490K:I538K and Q486E:I499L (also known as EL:KK). In one domain glutamic acid (E) at position 490 and isoleucine (I) at position 538 were replaced with lysine (K) (E490K:I538K), in partner domain glutamine (Q) at position 486 was replaced with glutamic acid and isoleucine at position 499 was replaced with leucine (L) (Q486E:I499L). As lysine is positively charged amino acid, variants with two additional lysine residues were referred to as ‘+’ and the other variants were referred to as ‘-‘, due to negative charge of introduced glutamic acid. Model of how variants may interact with each other is shown in [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F2.html Figure 2e]. Variants ‘+’ and ‘-‘ exhibited strong preference for heterodimerization and very weak preference for homodimerization when connected with suitable ZFP (L or R ZFP) in GFP reporter system. In [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F2.html Figure 2f] we can see that double mutations heterodimers (marked as L+/R- and L-/R+) have higher efficiency for gene correction than wild-type (wt) dimer (marked as Lwt/Rwt) and homodimers of double mutation variants (marked as L+/R+ and L-/R-) have almost none. Results of GFP reporter system indicate that formation of obligated heterodimer was achieved<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>.

=== Validation of heterodimeric FokI cleavage domain variants ===
==== Efficient endogenous gene editing ====
To confirm that the heterodimer variants preserved catalytic function researchers fused them with ZFPs that target a sequence present in exon 5 of the human gene for interleukin-2 receptor γ-chain (IL2Rγ). Designed ZFNs induced gene editing that introduced novel BsrBI restriction site in IL2Rγ [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F3.html (Figure 3a)]. Restriction enzyme BsrBI is able to cut at such restriction site resulting in additional band on electrophoresis. Results of gene editing efficiency shown in [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F3.html Figure 3b] indicate BsrBI restriction site was successfully inserted in case of ZFN with wt cleavage domain (Lwt/Rwt) and both heterodimeric combinations (L+/R- and L-/R+). Forced homodimers (L+/R+ and L-/R-) yielded no detectable gene editing. These results confirmed obligated heterodimerization of developed variants<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>.

==== Suppression of homodimer function ''in vitro'' ====
Cleavage activity ''in vitro'' was directly measured by radiolabeling of target sequences for ZFNs [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F4.html (Figure 4a)]. Target sequences were then digested by ZFNs with wt cleavage domain and variants of domain with double mutations, both in heterodimeric and homodimeric form. Migration of cleaved and uncleaved products in [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F4.html Figure 4b] indicate that wt cleavage domain (Lwt/Rwt) was active in heterodimeric and homomeric form, while efficient cleavage with double mutation variants (L+/R- and L-/R+) was only observed if domains formed heterodimer. This result once again supports development of variants, which form obligated heterodimer<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>.

==== Reduced levels of genome-wide cleavage ====
The aim of study was to develop more specific ZFNs that do not cleave off-target. Therefore, researchers conducted the next experiment on ZFN specificity of gene modification in mammalian cells. ZFN cleavage was determined by detection of proteins that localise at DNA DSBs. The proteins bound were detected by antibody-mediated technique. Protein that localise at DNA damage site and form foci, is tumour suppressor p63-binding protein 1 (53BP1). Protein 53BP1 was detected by immunofluorescence in target cells, which were transfected with ZFN expression constructs and treated with anti-53BP1 rabbit polyclonal antibodies. Results in [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F5.html Figure 5a] show that signal is weaker in cells transfected with ZFN with double mutations variants compared to ZFN with wt cleavage domain. This indicates cleavage activity was reduced in case of transfection with modified ZFN. Western blot analysis [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F5.html (Figure 5b)] confirmed that both ZFNs were expressed at comparable levels, suggesting that the reduced activity was a result of decreased off-target effect. Alternative damage marker is phosphorylated histone H2AX (γH2AX), which also forms foci at DSBs. Flow cytometry data [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F6.html (Figure 6a)] for target cells transfected with ZFNs and stained with antibodies against γH2AX showed that number of positive cells (cells forming γH2AX foci) was significantly lower for ZFNs with double mutations variants compared to ZFN with wt cleavage domain. In addition gene modification was monitored by Surveyor Nuclease assay, which determines the frequency of the small insertions and deletions (indels) characteristic of unspecific DSB repair by NHEJ. Results [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F6.html (Figure 6b)] showed comparable levels of gene modification in cells treated with ZFNs with wt and double mutations cleavage domains, indicating comparable cellular activities at target locus. These results confirmed that heterodimeric FokI cleavage domain variants retain catalytic activity while exhibiting reduced levels of off-target cleavage<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>.

=== Conclusion ===
In conclusion, Miller and colleagues developed two complementary FokI cleavage domain variants with double mutation, which function as an obligate heterodimer and improve ZFN specificity limiting off-target effect. Researchers predicted that their new architecture of FokI cleavage domain could be beneficial for therapeutic application of ZFNs due to additional mechanism for reducing off-target cleavage. It could also be helpful for less safety-intensive application such as crop engineering, cell-line engineering and construction of disease models. These ZFNs would be useful for gene modification protocols requiring simultaneous cleavage at multiple targets, where it would eliminate unwanted combination of ZFNs<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>.

== Further research ==
In 2011 same research group form Sangamo BioSciences published a [http://www.nature.com/nmeth/journal/v8/n1/full/nmeth.1539.html new paper] in Nature Methods discussing improved obligate heterodimeric architectures. In this study yeast-based selection system was used to cross-examine the dimer interface. A reporter assay was used to isolate FokI with mutations causing cold-sensitivity, phenotype resulting in diminished cleavage activity at lower temperatures but still be active at higher ones. The hypothesis of study was that cold-sensitive mutants would affect critical residues involved in dimerization, because this class of mutations is often associated with incorrect assembly of multisubunit protein complexes<ref name="ref10">Doyon, Y.; Vo, T.D.; Mendel, M.C.; Greenberg, S.G.; Wang, J.; Xia, D.F.; Miller, J.C.; Urnov, F.D.; Gregory, P.D.; Holmes, M.C. Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures. ''Nat Methods''., 2011, 8(1), 74-79.</ref>.

A selection system in ''Saccharomyces cerecisiae'' developed to isolate ZFN mutants with cold-sensitivity phenotype included two independent single-stranded annealing reporter constructs that were integrated into the yeast genome. DNA sequence for reporter genes MEL1 and PHO5 were interrupted by binding site for ZFN homodimer (CCR5-L) [http://www.nature.com/nmeth/journal/v8/n1/fig_tab/nmeth.1539_F1.html (Figure 1a)]. Additionally PHO5 reporter system had incorporated positive selection cassette natMX that enables resistance to antibiotic neuroseothricin. MEL1 reporter system contained the URA3 gene for negative selection using 5-fluoroorotic acid, which can be converted into the toxic compound causing cell death and kanMX cassette that enables resistance to antibiotic geneticin. ZFN-induced DSB would result in restoration of reporter gene expression and simultaneous elimination of all positive and negative selection markers. Library of FokI mutants was constructed by error-prone PCR that randomly mutagenized the sequence [http://www.nature.com/nmeth/journal/v8/n1/fig_tab/nmeth.1539_F1.html (Figure 1b)]. ZFN expression was induced at 22 °C, and then cells were collected and incubated in medium with geneticin and neurseothricin. With this step all cells carrying active ZFN constructs were eliminated. Remaining cells were shifted to 37 °C and plated on medium with 5-fluoroorotic acid and colorimetric substrate for Pho5. This step gave them conditionally active ZFNs. Constructs were then isolated and tested at 22 °C, 30 °C and 37 °C to confirm cold-sensitive phenotype [http://www.nature.com/nmeth/journal/v8/n1/fig_tab/nmeth.1539_F1.html (Figure 1c)]; 16 mutants had minimal activity at 22 °C but had restored reporter gene expression at higher temperature. Within isolated mutants previously altered residues that contributed to obligated heterodimerization (glutamine 486 and isoleucine 499 and 538) were found. Their focus was residues asparagine 496 and histidine 537, which face each other in dimerization interface. These two residues were substituted with a pair of oppositely charged amino acids into EL:KK FokI backbone. This substitution could strengthen dimerization in the context of the obligate heterodimer variants. The asparagine 496 was replaced with negatively charged glutamic and aspartic acid in the EL domain, and histidine 537 was replaced with positively charged lysine and arginine in the KK domain. Measured cleavage activity comparted to both wt and EL:KK versions suggested that aspartic acid 496 drove maximal activity in EL monomer, whereas lysine and arginine at position 537 resulted in similar activity improvements. This domains were referred to as ELD:KKK or ELD:KKR. Improved activity of new FokI variants was confirmed by various assays. All results supported superior cleavage activity and retainment of obligate heterodimer function. Researchers concluded that these enhanced FokI domains were portable to many ZFPs, independent of cell type and are a general solution for improved ZFN activity<ref name="ref10">Doyon, Y.; Vo, T.D.; Mendel, M.C.; Greenberg, S.G.; Wang, J.; Xia, D.F.; Miller, J.C.; Urnov, F.D.; Gregory, P.D.; Holmes, M.C. Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures. ''Nat Methods''., 2011, 8(1), 74-79.</ref>.

==References==
<references />

[[SB students resources]]

An improved zinc-finger nuclease architecture for highly specific genome editing

2015-01-11T12:41:10Z

EvaKnapič:

Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J.[http://www.nature.com/nbt/journal/v25/n7/full/nbt1319.html An improved zinc-finger nuclease architecture for highly specific genome editing]. ''Nat Biotechnol''., 2007, 25(7), 778-785.

(Eva Knapič)

== Genome editing ==
Genome editing is a type of genetic engineering in which DNA is inserted, removed or replaced from genome using artificially designed nucleases, enzymes that cleave the phosphodiester bonds between nucleotides. The principle of method is based on nuclease cleavage at a desired site of the genome in order to create specific double-stranded break, then put to use the cell’s endogenous mechanisms to repair the induced break by homology-directed repair or non-homologous end joining. There are a few different classes of engineered nucleases that are currently being used: zinc-finger nucleases, transcription activator-like effector nucleases and the CRISPR/Cas system. In this seminar we will focus on zinc finger nucleases. Firstly, we will take a look at basics of zinc-finger nucleases and their mechanism. Then we will discuss improved zinc-finger nuclease architecture for more specific efficiency described by Miller and colleagues in their paper published in Nature Biotechnology in 2007<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>.

== Zinc-finger nuclease ==
Zinc-finger nuclease (ZFN) is an artificially designed endonuclease that can be customised to cleave at sequence specific site on the DNA. The enzyme consists of two domains: DNA-binding domain composed of zinc finger structural motifs and DNA-cleavage domain of restriction enzyme FokI. Domains are connected with short inter-domain linker. This structure combines key qualities of DNA binding specificity, flexibility of zinc-finger motifs and cleavage activity of FokI catalytic domain that is robust but restricted. Furthermore all three elements can be optimised for retargeting and improving efficiency<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref><ref name="ref2">Urnov, F. D.; Rebar, E.J.; Holmes, M. C.; Zhang, H. S.; Gregory, P.D. Genome editing with engineered zinc finger nucleases. ''Nat Rev Genet''., 2010, 11(9), 636-646.</ref>.

=== DNA-binding domain ===
As mentioned above, zinc finger motifs represent DNA-binding domain of ZFN. [http://commons.wikimedia.org/wiki/File:Zinc_finger_rendered.png#mediaviewer/File:Zinc_finger_rendered.png Zinc fingers] are small protein structural motifs distinguished by coordinated zinc ions that stabilise the protein fold. Each zinc-finger recognises 3 base pairs (bp) of DNA. Contacts between the zinc fingers and DNA are made by α-helix that binds in the DNA major groove<ref name="ref3">Zinc finger ; Wikipedia the free encylopedia [cited 2.1.2015]. http://en.wikipedia.org/wiki/Zinc_finger.</ref>. Proteins that contain any number of zinc finger motifs are called zinc-finger proteins (ZFPs). Engineered ZFPs consist of three to six zinc finger motifs thus enabling binding of 9 bp to 18 bp targets. Longer recognition sequence improves specificity and precision of ZFPs. ZFP based DNA-binding domains can be coupled to various effector domains, which then cuts the DNA sequence determined by the ZFP<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref><ref name="ref2">Urnov, F. D.; Rebar, E.J.; Holmes, M. C.; Zhang, H. S.; Gregory, P.D. Genome editing with engineered zinc finger nucleases. ''Nat Rev Genet''., 2010, 11(9), 636-646.</ref>.

There are multiple approaches to designing new ZFPs with binding specificities chosen by user. The simplest and widely used method to generate new zinc finger sets is modular assembly. Candidate ZFPs for target sequence are obtained by determining individual zinc fingers that bind each triplet and assembling them together thus recognising the target sequence<ref name="ref2">Urnov, F. D.; Rebar, E.J.; Holmes, M. C.; Zhang, H. S.; Gregory, P.D. Genome editing with engineered zinc finger nucleases. ''Nat Rev Genet''., 2010, 11(9), 636-646.</ref>. Alternative methods for designing new ZFPs include two-finger modules, oligomerised pool engineering (OPEN) and Context-Dependent Assembly (CoDA). In two-finger modules instead of individual fingers, construct of two-fingers is used for recognising target DNA sequence. Advantage of this approach is better optimisation of finger junctions and speed of finding and assembling suitable zinc fingers, a limitation is extension of initial characterisation of two-finger units <ref name="ref4">Carroll, D. Genome engineering with zinc-finger nucleases. ''Genetics''., 2011, 188(4), 773-782.</ref>. The OPEN system approach is carried out in two steps, firstly appropriate finger pools are recombined to create small combinatorial library, then members of library that efficiently bind to the target site are isolated using bacterial two-hybrid selection method in which binding of a zinc finger domain to its target activates expression of selected marker gene<ref name="ref5">Maeder M. L. et al. Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification. ''Mol Cell''., 2008, 31(2), 294–301.</ref>. CoDA is publicly available platform of reagents and software. With this approach three-finger sets are assembled using N- and C- terminal fingers that have been previously identified in other arrays with common middle finger. CoDA does not treat fingers as independent modules but instead explicitly accounts for context-dependent effects between adjacent fingers, increasing the probability of efficiency <ref name="ref6">Sander J.D. et al. Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA). ''Nat Methods''., 2011, 8(1), 67-69.</ref>. Regardless of which approach is used for designing new ZFPs ''in vitro'', their application in living cells is not always successful. Reason is the complexity of genome that often contains naturally occurring multiple copies of sequence that are either identical or very similar to target sequence and these copies can act as additional targets for ZFNs. Another complication is chromatin structure at target sites that may obstruct cleavage<ref name="ref4">Carroll, D. Genome engineering with zinc-finger nucleases. ''Genetics''., 2011, 188(4), 773-782.</ref>.

=== Inter-domain linker ===
Inter-domain linker connects DNA-binding domain with DNA-cleavage domain. Length of the linker is responsible for the shaping of spacer. Spacer is short sequence between both recognition sites for DNA-binding domains, which enables precise definition of cleavage site. Wilson and colleagues have demonstrated that sites with 5 bp spacer lengths showed the most efficient ZFN mediated gene targeting when the linker is 2 or 4 amino acids and that sites with 3 or 4 bp spacers could not be targeted efficiently. Group also demonstrated that ZFN variants with 5 amino acids linker can efficiently target sites with a 7 bp spacer. However, the ideal linker with corresponding spacer can only be determined by taking into consideration both ZFN activity on the chromosomal target site and ZFN associated toxicity <ref name="ref7">Wilson, K.A.; McEwen, A.E.; Pruett-Miller, S.M.; Zhang, J.; Kildebeck, E.J.;, Porteus, M.H. Expanding the Repertoire of Target Sites for Zinc Finger Nuclease-mediated Genome Modification. Mol Ther Nucleic Acids., 2013, 2, e88.</ref>.

=== DNA-cleavage domain ===
DNA-cleavage domain of ZFN originates from FokI catalytic domain. FokI is type IIS restriction endonuclease consisting of an N-terminal DNA-binding domain and non-specific DNA-cleavage C-terminal domain. In order to cleave DNA two FokI catalytic domains must form dimer. Each subunit contains one catalytic centre that is responsible for cleaving one strand of DNA duplex. This characteristic of FokI catalytic domain is crucial for successful genome editing with ZFN. Dimerization requires two independent binding events, which must occur in correct orientation and spacing between monomers. DNA-cleavage domain is usually fused to the C-terminus of DNA-binding domain, thus two individual ZFNs must bind to opposite strands of DNA. This enables specific targeting of long and potentially unique recognition sites<ref name="ref2">Urnov, F. D.; Rebar, E.J.; Holmes, M. C.; Zhang, H. S.; Gregory, P.D. Genome editing with engineered zinc finger nucleases. ''Nat Rev Genet''., 2010, 11(9), 636-646.</ref>.

== ZFN-mediated genome editing ==
ZFNs create double-stranded breaks (DSBs) in DNA at sequence specific site. DSBs can be repaired by cell’s endogenous mechanisms: homology-directed repair (HDR) or non-homologous end joining (NHEJ). There are few different possible outcomes of DSB repair. If the break is repaired by NHEJ, it can lead to gene disruption, tag ligation or large deletion. Gene disruption occurs when two broken ends are ligated back together that can lead to small insertions or deletions at the break site causing target gene disruption. Tag ligation occurs when adaptor with sticky ends is provided and is ligated into the chromosome producing tagged allele. When two different ZFNs cleave simultaneous same chromosome, entire segment between them is deleted. If the break is repaired by HDR, which occurs in the presence of donor DNA, it can result in gene correction, targeted gene addition or transgene stacking. If donor with small change, for example single bp, is provided, editing of endogenous allele results in gene correction. This enables easier functional genomics studies, modelling of disease caused by point mutation and gene therapy research. If provided donor carries transgene and has specific homology arms, transgene can be efficiently integrated into chromosome at break site, which results in targeted gene addition. Transgene stacking occurs when provided donor carries multiple linked transgenes between the homology arms. With this approach not only gene addition can be done, but also gene correction if DSB is created within or near gene in question and provided donor carries modified gene sequence<ref name="ref2">Urnov, F. D.; Rebar, E.J.; Holmes, M. C.; Zhang, H. S.; Gregory, P.D. Genome editing with engineered zinc finger nucleases. ''Nat Rev Genet''., 2010, 11(9), 636-646.</ref><ref name="ref4">Carroll, D. Genome engineering with zinc-finger nucleases. ''Genetics''., 2011, 188(4), 773-782.</ref><ref name="ref8">Gaj, T.; Gersbach, C.A.; Barbas, C.F. 3rd. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. ''Trends Biotechnol''., 2013, 31(7), 397-405.</ref>.

=== Applications: ===
==== Model organisms ====
ZFNs enable introduction of targeted modification in several model organisms used in common biological researches. Benefits of DSB repaired by cell’s endogenous mechanisms include: simple production of knock-out or knock-in cell lines, permanent and heritable mutation, compatibility with variety of species<ref name="ref8">Gaj, T.; Gersbach, C.A.; Barbas, C.F. 3rd. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. ''Trends Biotechnol''., 2013, 31(7), 397-405.</ref>.
==== Therapeutic application of ZFNs ====
ZFN-mediated gene modification by HDR has been used to correct mutations associated with X-linked severe combined immune deficiency (SCID), haemophilia B, sickle-cell disease and α1-antitrypsin deficiency. ZFN induced gene targeting by NHEJ repair has been shown as potential treatment for HIV/AIDS, either by targeting cellular co-receptors for HIV in primary T cells and hematopoietic stem/progenitor cells (clinical trials) or by targeting proviral HIV DNA<ref name="ref8">Gaj, T.; Gersbach, C.A.; Barbas, C.F. 3rd. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. ''Trends Biotechnol''., 2013, 31(7), 397-405.</ref>.

=== Potential problems ===
If the DNA-binding domain is not specific enough or target site is not unique in genome, ZFN may cleave at undesired location in the genome. Another potential problem is formation of ZFN homodimers that are by products of ZFN heterodimer that can cleave off-target. Besides cleaving off-target, ZFN homodimers mediate toxic effect. Undesired cleavage can lead to unwanted insertions or deletions at additional cleavage sites or vast number of DSBs can cause cell death. This can be avoided by extensive bioinformatics research beforehand, construction of ZFP that recognises longer and consequently rarer target sequences, usingf suitable linkers and cleavage domains that do not form unwanted homodimers<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref><ref name="ref2">Urnov, F. D.; Rebar, E.J.; Holmes, M. C.; Zhang, H. S.; Gregory, P.D. Genome editing with engineered zinc finger nucleases. ''Nat Rev Genet''., 2010, 11(9), 636-646.</ref>.

== AN IMPROVED ZINC-FINGER NUCLEASE ARCHITECTURE ==
Miller and colleagues present in their paper an improved ZFN architecture for more specific genome targeting. Their aim was to develop FokI cleavage domain variants that function as obligate heterodimers<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>.

=== Development of heterodimeric FokI cleavage domain variants ===
There are few things that need to be put in consideration when altering architecture of FokI DNA-cleavage domain. Firstly, FokI is an enzyme, this means goal is to improve interaction between domains but at the same time, catalytic function of domains must be conserved. Secondly, interaction between FokI DNA-cleavage domains in dimer has very low affinity, which is essential for ensuring cleavage specificity. Lastly, interface of FokI dimer is hydrophilic. Considering all the facts, Miller and colleagues developed strategy that involved step by step approach to modification of dimer interface coupled with direct testing of candidate variants for catalytic activity. Reporter system with green fluorescent protein (eGFP) was used to detect gene correction activity. The system uses HEK293 cell line containing a copy of the eGFP gene that is disrupted by short DNA fragment and has characterised ZFN target sequence [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F2.html (Figure 2a)]. Screening is based on ZFN cleavage of DNA fragment that is corrected by HDR using exogenous donor DNA containing missing eGFP sequence as template. If repair is successful, green fluorescence can be detected by fluorescence-activated cell sorting [http://commons.wikimedia.org/wiki/File:Fluorescence_Assisted_Cell_Sorting_(FACS)_B.jpg#mediaviewer/File:Fluorescence_Assisted_Cell_Sorting_(FACS)_B.jpg (FACS)]<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>. FACS is specialized type of flow cytometry, method for separating heterogeneous mixture of biological cells, which is based upon the specific light scattering at fluorescent wavelength<ref name="ref9">Flow cytometry; Wikipedia the free encylopedia [cited 2.1.2015]. http://en.wikipedia.org/wiki/Flow_cytometry#Fluorescence-activated_cell_sorting_.28FACS.29.</ref>. Results are shown in [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F2.html Figure 2b], left for cells transfected with donor DNA only and right for cells transfected with donor DNA and ZFNs. Step by step modification of interaction surface was carried out in four cycles. Modifications were implemented alternately, which means in each cycle only one partner domain was modified. Mutations were designed to form charge-charge interaction. Each variant was tested for gene correction activity as a heterodimer with unmodified partner ZFN and as a homodimer. More in detailed view of introduced mutation can be found in [http://www.nature.com/nbt/journal/v25/n7/suppinfo/nbt1319_S1.html Supplementary table 1]. In each cycle researchers identified a variant of domain that induced gene correction as a heterodimer and at the same time the domain had reduced activity as a homodimer [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F2.html (Figure 2d)]. Variants with best gene correction rate contained double mutations E490K:I538K and Q486E:I499L (also known as EL:KK). In one domain glutamic acid (E) at position 490 and isoleucine (I) at position 538 were replaced with lysine (K) (E490K:I538K), in partner domain glutamine (Q) at position 486 was replaced with glutamic acid and isoleucine at position 499 was replaced with leucine (L) (Q486E:I499L). As lysine is positively charged amino acid, variants with two additional lysine residues were referred to as ‘+’ and the other variants were referred to as ‘-‘, due to negative charge of introduced glutamic acid. Model of how variants may interact with each other is shown in [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F2.html Figure 2e]. Variants ‘+’ and ‘-‘ exhibited strong preference for heterodimerization and very weak preference for homodimerization when connected with suitable ZFP (L or R ZFP) in GFP reporter system. In [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F2.html Figure 2f] we can see that double mutations heterodimers (marked as L+/R- and L-/R+) have higher efficiency for gene correction than wild-type (wt) dimer (marked as Lwt/Rwt) and homodimers of double mutation variants (marked as L+/R+ and L-/R-) have almost none. Results of GFP reporter system indicate that formation of obligated heterodimer was achieved<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>.

=== Validation of heterodimeric FokI cleavage domain variants ===
==== Efficient endogenous gene editing ====
To confirm that the heterodimer variants preserved catalytic function researchers fused them with ZFPs that target a sequence present in exon 5 of the human gene for interleukin-2 receptor γ-chain (IL2Rγ). Designed ZFNs induced gene editing that introduced novel BsrBI restriction site in IL2Rγ [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F3.html (Figure 3a)]. Restriction enzyme BsrBI is able to cut at such restriction site resulting in additional band on electrophoresis. Results of gene editing efficiency shown in [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F3.html Figure 3b] indicate BsrBI restriction site was successfully inserted in case of ZFN with wt cleavage domain (Lwt/Rwt) and both heterodimeric combinations (L+/R- and L-/R+). Forced homodimers (L+/R+ and L-/R-) yielded no detectable gene editing. These results confirmed obligated heterodimerization of developed variants<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>.

==== Suppression of homodimer function ''in vitro'' ====
Cleavage activity ''in vitro'' was directly measured by radiolabeling of target sequences for ZFNs [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F4.html (Figure 4a)]. Target sequences were then digested by ZFNs with wt cleavage domain and variants of domain with double mutations, both in heterodimeric and homodimeric form. Migration of cleaved and uncleaved products in [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F4.html Figure 4b] indicate that wt cleavage domain (Lwt/Rwt) was active in heterodimeric and homomeric form, while efficient cleavage with double mutation variants (L+/R- and L-/R+) was only observed if domains formed heterodimer. This result once again supports development of variants, which form obligated heterodimer<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>.

==== Reduced levels of genome-wide cleavage ====
The aim of study was to develop more specific ZFNs that do not cleave off-target. Therefore, researchers conducted the next experiment on ZFN specificity of gene modification in mammalian cells. ZFN cleavage was determined by detection of proteins that localise at DNA DSBs. The proteins bound were detected by antibody-mediated technique. Protein that localise at DNA damage site and form foci, is tumour suppressor p63-binding protein 1 (53BP1). Protein 53BP1 was detected by immunofluorescence in target cells, which were transfected with ZFN expression constructs and treated with anti-53BP1 rabbit polyclonal antibodies. Results in [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F5.html Figure 5a] show that signal is weaker in cells transfected with ZFN with double mutations variants compared to ZFN with wt cleavage domain. This indicates cleavage activity was reduced in case of transfection with modified ZFN. Western blot analysis [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F5.html (Figure 5b)] confirmed that both ZFNs were expressed at comparable levels, suggesting that the reduced activity was a result of decreased off-target effect. Alternative damage marker is phosphorylated histone H2AX (γH2AX), which also forms foci at DSBs. Flow cytometry data [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F6.html (Figure 6a)] for target cells transfected with ZFNs and stained with antibodies against γH2AX showed that number of positive cells (cells forming γH2AX foci) was significantly lower for ZFNs with double mutations variants compared to ZFN with wt cleavage domain. In addition gene modification was monitored by Surveyor Nuclease assay, which determines the frequency of the small insertions and deletions (indels) characteristic of unspecific DSB repair by NHEJ. Results [http://www.nature.com/nbt/journal/v25/n7/fig_tab/nbt1319_F6.html (Figure 6b)] showed comparable levels of gene modification in cells treated with ZFNs with wt and double mutations cleavage domains, indicating comparable cellular activities at target locus. These results confirmed that heterodimeric FokI cleavage domain variants retain catalytic activity while exhibiting reduced levels of off-target cleavage<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>.

=== Conclusion ===
In conclusion, Miller and colleagues developed two complementary FokI cleavage domain variants with double mutation, which function as an obligate heterodimer and improve ZFN specificity limiting off-target effect. Researchers predicted that their new architecture of FokI cleavage domain could be beneficial for therapeutic application of ZFNs due to additional mechanism for reducing off-target cleavage. It could also be helpful for less safety-intensive application such as crop engineering, cell-line engineering and construction of disease models. These ZFNs would be useful for gene modification protocols requiring simultaneous cleavage at multiple targets, where it would eliminate unwanted combination of ZFNs<ref name="ref1">Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J. An improved zinc-finger nuclease architecture for highly specific genome editing. ''Nat Biotechnol''., 2007, 25(7), 778-785.</ref>.

== Further research ==
In 2011 same research group form Sangamo BioSciences published a [http://www.nature.com/nmeth/journal/v8/n1/full/nmeth.1539.html new paper] in Nature Methods discussing improved obligate heterodimeric architectures. In this study yeast-based selection system was used to cross-examine the dimer interface. A reporter assay was used to isolate FokI with mutations causing cold-sensitivity, phenotype resulting in diminished cleavage activity at lower temperatures but still be active at higher ones. The hypothesis of study was that cold-sensitive mutants would affect critical residues involved in dimerization, because this class of mutations is often associated with incorrect assembly of multisubunit protein complexes<ref name="ref10">Doyon, Y.; Vo, T.D.; Mendel, M.C.; Greenberg, S.G.; Wang, J.; Xia, D.F.; Miller, J.C.; Urnov, F.D.; Gregory, P.D.; Holmes, M.C. Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures. ''Nat Methods''., 2011, 8(1), 74-79.</ref>.

A selection system in ''Saccharomyces cerecisiae'' developed to isolate ZFN mutants with cold-sensitivity phenotype included two independent single-stranded annealing reporter constructs that were integrated into the yeast genome. DNA sequence for reporter genes MEL1 and PHO5 were interrupted by binding site for ZFN homodimer (CCR5-L) [http://www.nature.com/nmeth/journal/v8/n1/fig_tab/nmeth.1539_F1.html (Figure 1a)]. Additionally PHO5 reporter system had incorporated positive selection cassette natMX that enables resistance to antibiotic neuroseothricin. MEL1 reporter system contained the URA3 gene for negative selection using 5-fluoroorotic acid, which can be converted into the toxic compound causing cell death and kanMX cassette that enables resistance to antibiotic geneticin. ZFN-induced DSB would result in restoration of reporter gene expression and simultaneous elimination of all positive and negative selection markers. Library of FokI mutants was constructed by error-prone PCR that randomly mutagenized the sequence [http://www.nature.com/nmeth/journal/v8/n1/fig_tab/nmeth.1539_F1.html (Figure 1b)]. ZFN expression was induced at 22 °C, and then cells were collected and incubated in medium with geneticin and neurseothricin. With this step all cells carrying active ZFN constructs were eliminated. Remaining cells were shifted to 37 °C and plated on medium with 5-fluoroorotic acid and colorimetric substrate for Pho5. This step gave them conditionally active ZFNs. Constructs were then isolated and tested at 22 °C, 30 °C and 37 °C to confirm cold-sensitive phenotype [http://www.nature.com/nmeth/journal/v8/n1/fig_tab/nmeth.1539_F1.html (Figure 1c)]; 16 mutants had minimal activity at 22 °C but had restored reporter gene expression at higher temperature. Within isolated mutants previously altered residues that contributed to obligated heterodimerization (glutamine 486 and isoleucine 499 and 538) were found. Their focus was residues asparagine 496 and histidine 537, which face each other in dimerization interface. These two residues were substituted with a pair of oppositely charged amino acids into EL:KK FokI backbone. This substitution could strengthen dimerization in the context of the obligate heterodimer variants. The asparagine 496 was replaced with negatively charged glutamic and aspartic acid in the EL domain, and histidine 537 was replaced with positively charged lysine and arginine in the KK domain. Measured cleavage activity comparted to both wt and EL:KK versions suggested that aspartic acid 496 drove maximal activity in EL monomer, whereas lysine and arginine at position 537 resulted in similar activity improvements. This domains were referred to as ELD:KKK or ELD:KKR. Improved activity of new FokI variants was confirmed by various assays. All results supported superior cleavage activity and retainment of obligate heterodimer function. Researchers concluded that these enhanced FokI domains were portable to many ZFPs, independent of cell type and are a general solution for improved ZFN activity<ref name="ref10">Doyon, Y.; Vo, T.D.; Mendel, M.C.; Greenberg, S.G.; Wang, J.; Xia, D.F.; Miller, J.C.; Urnov, F.D.; Gregory, P.D.; Holmes, M.C. Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures. ''Nat Methods''., 2011, 8(1), 74-79.</ref>.

==References==
<references />

[[SB students resources]]

An improved zinc-finger nuclease architecture for highly specific genome editing

2015-01-11T12:33:12Z

EvaKnapič:

An improved zinc-finger nuclease architecture for highly specific genome editing

2015-01-11T12:05:04Z

EvaKnapič:

An improved zinc-finger nuclease architecture for highly specific genome editing

2015-01-11T11:52:20Z

EvaKnapič: New page: Miller, J.C.; Holmes, M.C.; Wang, J.; Guschin, D.Y.; Lee, Y.L.; Rupniewski, I.; Beausejour, C.M.; Waite, A.J.; Wang, N.S.; Kim, K.A.; Gregory, P.D.; Pabo, C.O.; Rebar, E.J.[http://www.natu...

Automated Multiplexing Quantum Dots in Situ Hybridization Assay

2013-12-09T08:13:40Z

EvaKnapič:

Povzeto po: Zhang W, Hubbard A, Brunhoeber P, Wang Y, Tang L. Automated Multiplexing Quantum Dots in Situ Hybridization Assay for Simultaneous Detection of ERG and PTEN Gene Status in Prostate Cancer. ''J Mol Diagn'', 2013, 15(6), str. 754-764.

<h2>UVOD</h2>

Rak prostate je ena najpogostejših oblik rakavih obolenj pri moških. Pomembna dejavnika, ki vplivata na razvoj raka prostate sta starost in družinska obremenjenost. V raziskavi predstavljeni v članku, so preverjali uporabo kvantnih točk (QD – "quantum dots") pri razvoju avtomatiziranega multipleksnega testa ''in situ'' hibridizacije (ISH) za detekcijo genov ERG in PTEN v vzorcih pripravljenih iz tkiva prostate. QD so polprevodniški nanokristali z ozkim emisijskim spektrom, ki olajša sočasno detekcijo več celičnih tarč. So zelo svetli in so manj nagnjeni k bledenju kot organski fluorofori, kar omogoča izboljšanje razmerja med signalom in šumom. ERG je onkogen, ki se prepiše v protein ERG, ta pa deluje kot transkripcijski regulator. PTEN je tumor supresorski gen, ki se prepiše v fosfatazni in tenzinski homolog (PTEN), ki je udeležen pri regulaciji celičnega cikla, kjer preprečuje prehitro celično rast in delitev celic. Preureditev ERG in delecija PTEN sta najpogostejši genomski spremembi pri raku prostate.

<h2>EKSPERIMENTALNI DEL</h2>

Za delo so uporabili avtomatski štiribarven multipleksen QD ISH test, ki omogoča detekcijo klinično pomembnih biomarkerjev raka prostate. Pridobili so deset vzorcev tkiva prostate, od tega je bilo osem vzorcev diagnosticiranih kot benigno tkivo, dva pa kot rak prostate. Razvili so štiri DNA sonde: ERG3p, ERG5p, PTEN in CEN10. Sondi ERG3p in ERG5p sta bili oblikovani tako, da ciljata centromerno ter telomerno regijo na kromosomu 21, kjer se nahaja ERG. Sondo ERG5p so označili z DIG, ERG3p sondo pa so označili z DNP. Sondo PTEN so oblikovali tako, da je ciljala regijo kromosoma 10, na kateri se nahaja PTEN, označili pa so jo s TS. Sondo CEN10 so pridobili s pomočjo plazmida pA10RP8, ki cilja na centromero kromosoma 10, označili so jo z NP. Za detekcijo so uporabili štiri anti-haptenska protitelesa konjugirana s QD655, ki sveti rdeče, QD565, ki sveti zeleno, QD605, ki sveti roza in QD525, ki sveti modro.

<h3>Avtomatska štiribarvna ERG/PTEN QD ''in situ'' hibridizacija</h3>

Po pred obdelavi, so vzorcem dodali vse štiri DNA sonde. Denaturaciji genomske DNA in DNA sond pri 85°C za 8 minut, je sledila hibridizacija sond, ki je trajala 6 ur pri 44°C. Po treh spiranjih pri 72°C za 8 minut, so vzorcem dodali raztopino protiteles konjugiranimi s QD ter vse skupaj inkubirali 60 minut pri 37°C. Po trikratnem spiranju vzorcev z reakcijskim pufrom, so dodali DAPI.

<h2>REZULTATI</h2>

Ovrednotili so zmogljivost štiribarvnega ERG/PTEN QD ISH testa pri optimalni pred obdelavi vzorcev. Signale so prešteli pri 389 vzorcih, ki so jih pridobili iz desetih tkiv prostate. Izkazalo se je, da je bilo 91% obarvanj sprejemljivih tako za ERG3p ter ERG5p, kot tudi za PTEN ter CEN10. Ne sprejemljivih je bilo samo 36 od 386 preparatov, od katerih je pri 28 prišlo do visokega ozadja pri QD655, pri osemih pa so bili signali prešibki oziroma jih sploh ni bilo.
Med preparati s sprejemljivim obarvanjem je bilo 280 takih, ki so jih pridobili iz osem vzorcev benignega tkiva prostate in 70 takih, ki so bili pridobljeni iz dveh vzorcev tkiv z rakom prostate. Naključno so izbrali 19 preparatov pripravljenih iz dveh benignih tkiv in dveh tkiv z rakom prostate, katerim so prešteli signale za ERG3p, ERG5p, PTEN in CEN10 na jedro celice v treh različnih dneh. Tako pridobljeni podatki so pokazali, da so rezultati obarvanja konstanti skozi vse dni štetja. Pri preparatih iz vzorca benignega tkiva se je vrednost pozitivnih celic z ERG preurejanjem gibala med 0 ter 6%, razmerje PTEN/CEN10 je bilo med 0,94 do 1,0. Pri preparatih iz drugega benignega tkiva pa se je procent pozitivnih celic gibal od 0% do 2%, razmerje signalov PTEN/CEN10 pa je bilo med 0,9 in 1,1. Pri preparatih pripravljenih iz tkiva z rakom prostate se je vrednost pozitivnih celic z ERG preurejanjem gibala med 78% do 90%, razmerje med signali PTEN/CEN10 je bilo med 0,8 in 0,9. Pri preparatih pripravljenih iz drugega tkiva raka prostate pa se je vrednost pozitivnih celic gibala med 74% ter 88%, razmerje signalov PTEN/CEN10 pa je bilo 0,0.

<h2>ZAKLJUČEK</h2>

Štiribarven QD ISH test omogoča multipleksno detekcijo molekulskih biomarkerjev. Hkrati omogoča detekcijo štirih genomskih tarč na enem samem preparatu in tako poveča učinkovitost uporabe vzorca tkiva. Za samo interpretacijo signalov je potreben samo fluorescenčen mikroskop. Svetlost QD in njihova odpornost proti bledenju omogoča enostavno določevanje signalov. Popolna avtomatizacija testa pa omogoča hitro pridobitev rezultatov, ki so konstantni in natančni.

Automated Multiplexing Quantum Dots in Situ Hybridization Assay

2013-12-06T15:20:48Z

EvaKnapič:

Povzeto po: Zhang W, Hubbard A, Brunhoeber P, Wang Y, Tang L. Automated Multiplexing Quantum Dots in Situ Hybridization Assay for Simultaneous Detection of ERG and PTEN Gene Status in Prostate Cancer. ''J Mol Diagn'', 2013, 15(6), str. 754-764.

<h2>UVOD</h2>

Rak prostate je ena najpogostejših oblik rakavih obolenj pri moških. Pomembna dejavnika, ki vplivata na razvoj raka prostate sta starost in družinska obremenjenost. V raziskavi predstavljeni v članku, so preverjali uporabo kvantnih točk (QD – "quantum dots") pri razvoju avtomatiziranega multipleksnega testa ''in situ'' hibridizacije (ISH) za detekcijo genov ERG in PTEN v vzorcih pripravljenih iz tkiva prostate. QD so polprevodniški nanokristali z ozkim emisijskim spektrom, ki olajša sočasno detekcijo več celičnih tarč. So zelo svetli in so manj nagnjeni k bledenju kot organski fluorofori, kar omogoča izboljšanje razmerja med signalom in šumom. ERG je onkogen, ki se prepiše v protein ERG, ta pa deluje kot transkripcijski regulator. PTEN je tumor supresorski gen, ki se prepiše v fosfatazni in tenzinski homolog (PTEN), ki je udeležen pri regulaciji celičnega cikla, kjer preprečuje prehitro celično rast in delitev celic. Preureditev ERG in delecija PTEN sta najpogostejši genomski spremembi pri raku prostate.

<h2>EKSPERIMENTALNI DEL</h2>

Za delo so uporabili avtomatski štiribarven multipleksen QD ISH test, ki omogoča detekcijo klinično pomembnih biomarkerjev raka prostate. Pridobili so deset vzorcev tkiva prostate, od tega je bilo osem vzorcev diagnosticiranih kot benigno tkivo, dva pa kot rak prostate. Razvili so štiri DNA sonde: ERG3p, ERG5p, PTEN in CEN10. Sondi ERG3p in ERG5p sta bili oblikovani tako, da ciljata centromerno ter telomerno regijo na kromosomu 21, kjer se nahaja ERG. Sondo ERG5p so označili z DIG, ERG3p sondo pa so označili z DNP. Sondo PENT so oblikovali tako, da je ciljala regijo kromosoma 10, na kateri se nahaja PENT, označili pa so jo s TS. Sondo CEN10 so pridobili s pomočjo plazmida pA10RP8, ki cilja na centromero kromosoma 10, označili so jo z NP. Za detekcijo so uporabili štiri anti-haptenska protitelesa konjugirana s QD655, ki sveti rdeče, QD565, ki sveti zeleno, QD605, ki sveti roza in QD525, ki sveti modro.

<h3>Avtomatska štiribarvna ERG/PTEN QD ''in situ'' hibridizacija</h3>

Po pred obdelavi, so vzorcem dodali vse štiri DNA sonde. Denaturaciji genomske DNA in DNA sond pri 85°C za 8 minut, je sledila hibridizacija sond, ki je trajala 6 ur pri 44°C. Po treh spiranjih pri 72°C za 8 minut, so vzorcem dodali raztopino protiteles konjugiranimi s QD ter vse skupaj inkubirali 60 minut pri 37°C. Po trikratnem spiranju vzorcev z reakcijskim pufrom, so dodali DAPI.

<h2>REZULTATI</h2>

Ovrednotili so zmogljivost štiribarvnega ERG/PTEN QD ISH testa pri optimalni pred obdelavi vzorcev. Signale so prešteli pri 389 vzorcih, ki so jih pridobili iz desetih tkiv prostate. Izkazalo se je, da je bilo 91% obarvanj sprejemljivih tako za ERG3p ter ERG5p, kot tudi za PTEN ter CEN10. Ne sprejemljivih je bilo samo 36 od 386 preparatov, od katerih je pri 28 prišlo do visokega ozadja pri QD655, pri osemih pa so bili signali prešibki oziroma jih sploh ni bilo.
Med preparati s sprejemljivim obarvanjem je bilo 280 takih, ki so jih pridobili iz osem vzorcev benignega tkiva prostate in 70 takih, ki so bili pridobljeni iz dveh vzorcev tkiv z rakom prostate. Naključno so izbrali 19 preparatov pripravljenih iz dveh benignih tkiv in dveh tkiv z rakom prostate, katerim so prešteli signale za ERG3p, ERG5p, PTEN in CEN10 na jedro celice v treh različnih dneh. Tako pridobljeni podatki so pokazali, da so rezultati obarvanja konstanti skozi vse dni štetja. Pri preparatih iz vzorca benignega tkiva se je vrednost pozitivnih celic z ERG preurejanjem gibala med 0 ter 6%, razmerje PTEN/CEN10 je bilo med 0,94 do 1,0. Pri preparatih iz drugega benignega tkiva pa se je procent pozitivnih celic gibal od 0% do 2%, razmerje signalov PTEN/CEN10 pa je bilo med 0,9 in 1,1. Pri preparatih pripravljenih iz tkiva z rakom prostate se je vrednost pozitivnih celic z ERG preurejanjem gibala med 78% do 90%, razmerje med signali PTEN/CEN10 je bilo med 0,8 in 0,9. Pri preparatih pripravljenih iz drugega tkiva raka prostate pa se je vrednost pozitivnih celic gibala med 74% ter 88%, razmerje signalov PTEN/CEN10 pa je bilo 0,0.

<h2>ZAKLJUČEK</h2>

Štiribarven QD ISH test omogoča multipleksno detekcijo molekulskih biomarkerjev. Hkrati omogoča detekcijo štirih genomskih tarč na enem samem preparatu in tako poveča učinkovitost uporabe vzorca tkiva. Za samo interpretacijo signalov je potreben samo fluorescenčen mikroskop. Svetlost QD in njihova odpornost proti bledenju omogoča enostavno določevanje signalov. Popolna avtomatizacija testa pa omogoča hitro pridobitev rezultatov, ki so konstantni in natančni.

Automated Multiplexing Quantum Dots in Situ Hybridization Assay

2013-12-06T15:18:21Z

EvaKnapič: New page: Povzeto po: Zhang W, Hubbard A, Brunhoeber P, Wang Y, Tang L. Automated Multiplexing Quantum Dots in Situ Hybridization Assay for Simultaneous Detection of ERG and PTEN Gene Status in Pros...

Povzeto po: Zhang W, Hubbard A, Brunhoeber P, Wang Y, Tang L. Automated Multiplexing Quantum Dots in Situ Hybridization Assay for Simultaneous Detection of ERG and PTEN Gene Status in Prostate Cancer. J Mol Diagn, 2013, 15(6), str. 754-764.

<h2>UVOD</h2>

Rak prostate je ena najpogostejših oblik rakavih obolenj pri moških. Pomembna dejavnika, ki vplivata na razvoj raka prostate sta starost in družinska obremenjenost. V raziskavi predstavljeni v članku, so preverjali uporabo kvantnih točk (QD – "quantum dots") pri razvoju avtomatiziranega multipleksnega testa ''in situ'' hibridizacije (ISH) za detekcijo genov ERG in PTEN v vzorcih pripravljenih iz tkiva prostate. QD so polprevodniški nanokristali z ozkim emisijskim spektrom, ki olajša sočasno detekcijo več celičnih tarč. So zelo svetli in so manj nagnjeni k bledenju kot organski fluorofori, kar omogoča izboljšanje razmerja med signalom in šumom. ERG je onkogen, ki se prepiše v protein ERG, ta pa deluje kot transkripcijski regulator. PTEN je tumor supresorski gen, ki se prepiše v fosfatazni in tenzinski homolog (PTEN), ki je udeležen pri regulaciji celičnega cikla, kjer preprečuje prehitro celično rast in delitev celic. Preureditev ERG in delecija PTEN sta najpogostejši genomski spremembi pri raku prostate.

<h2>EKSPERIMENTALNI DEL</h2>

Za delo so uporabili avtomatski štiribarven multipleksen QD ISH test, ki omogoča detekcijo klinično pomembnih biomarkerjev raka prostate. Pridobili so deset vzorcev tkiva prostate, od tega je bilo osem vzorcev diagnosticiranih kot benigno tkivo, dva pa kot rak prostate. Razvili so štiri DNA sonde: ERG3p, ERG5p, PTEN in CEN10. Sondi ERG3p in ERG5p sta bili oblikovani tako, da ciljata centromerno ter telomerno regijo na kromosomu 21, kjer se nahaja ERG. Sondo ERG5p so označili z DIG, ERG3p sondo pa so označili z DNP. Sondo PENT so oblikovali tako, da je ciljala regijo kromosoma 10, na kateri se nahaja PENT, označili pa so jo s TS. Sondo CEN10 so pridobili s pomočjo plazmida pA10RP8, ki cilja na centromero kromosoma 10, označili so jo z NP. Za detekcijo so uporabili štiri anti-haptenska protitelesa konjugirana s QD655, ki sveti rdeče, QD565, ki sveti zeleno, QD605, ki sveti roza in QD525, ki sveti modro.

<h3>Avtomatska štiribarvna ERG/PTEN QD ''in situ'' hibridizacija</h3>

Po pred obdelavi, so vzorcem dodali vse štiri DNA sonde. Denaturaciji genomske DNA in DNA sond pri 85°C za 8 minut, je sledila hibridizacija sond, ki je trajala 6 ur pri 44°C. Po treh spiranjih pri 72°C za 8 minut, so vzorcem dodali raztopino protiteles konjugiranimi s QD ter vse skupaj inkubirali 60 minut pri 37°C. Po trikratnem spiranju vzorcev z reakcijskim pufrom, so dodali DAPI.

<h2>REZULTATI</h2>

Ovrednotili so zmogljivost štiribarvnega ERG/PTEN QD ISH testa pri optimalni pred obdelavi vzorcev. Signale so prešteli pri 389 vzorcih, ki so jih pridobili iz desetih tkiv prostate. Izkazalo se je, da je bilo 91% obarvanj sprejemljivih tako za ERG3p ter ERG5p, kot tudi za PTEN ter CEN10. Ne sprejemljivih je bilo samo 36 od 386 preparatov, od katerih je pri 28 prišlo do visokega ozadja pri QD655, pri osemih pa so bili signali prešibki oziroma jih sploh ni bilo.
Med preparati s sprejemljivim obarvanjem je bilo 280 takih, ki so jih pridobili iz osem vzorcev benignega tkiva prostate in 70 takih, ki so bili pridobljeni iz dveh vzorcev tkiv z rakom prostate. Naključno so izbrali 19 preparatov pripravljenih iz dveh benignih tkiv in dveh tkiv z rakom prostate, katerim so prešteli signale za ERG3p, ERG5p, PTEN in CEN10 na jedro celice v treh različnih dneh. Tako pridobljeni podatki so pokazali, da so rezultati obarvanja konstanti skozi vse dni štetja. Pri preparatih iz vzorca benignega tkiva se je vrednost pozitivnih celic z ERG preurejanjem gibala med 0 ter 6%, razmerje PTEN/CEN10 je bilo med 0,94 do 1,0. Pri preparatih iz drugega benignega tkiva pa se je procent pozitivnih celic gibal od 0% do 2%, razmerje signalov PTEN/CEN10 pa je bilo med 0,9 in 1,1. Pri preparatih pripravljenih iz tkiva z rakom prostate se je vrednost pozitivnih celic z ERG preurejanjem gibala med 78% do 90%, razmerje med signali PTEN/CEN10 je bilo med 0,8 in 0,9. Pri preparatih pripravljenih iz drugega tkiva raka prostate pa se je vrednost pozitivnih celic gibala med 74% ter 88%, razmerje signalov PTEN/CEN10 pa je bilo 0,0.

<h2>ZAKLJUČEK</h2>

Štiribarven QD ISH test omogoča multipleksno detekcijo molekulskih biomarkerjev. Hkrati omogoča detekcijo štirih genomskih tarč na enem samem preparatu in tako poveča učinkovitost uporabe vzorca tkiva. Za samo interpretacijo signalov je potreben samo fluorescenčen mikroskop. Svetlost QD in njihova odpornost proti bledenju omogoča enostavno določevanje signalov. Popolna avtomatizacija testa pa omogoča hitro pridobitev rezultatov, ki so konstantni in natančni.

Seminarji TehDNA

2013-10-08T14:13:25Z

EvaKnapič:

Seminarje iz Tehnologije DNA bo v študijskem letu 2013/14 vodila asist. dr. Helena Čelešnik.

Seznam tem za seminarje:

# Mutageneza (16.10.)
# Izražanje na površini (23.10.)
# Dvohibridni sistemi (30.10.)
# Mikromrežne tehnologije (6.11.)
# GSO v agronomiji (13.11.) Niki Bursič, Petra Malavašič
# Transgenske živali (27.11.) Andrea Grof, Eva Lucija Kozak
# Izvorne celice (4.12.) Sara Primec, Alja Zottel, Tjaša Goričan
# DNA-diagnostika (11.12.) Tina Gregorič , Eva Knapič, Veronika Jarc
# Forenzika, arheologija, sistematika (18.12.)
# Mikromreže, genomike (8.1.)
# Gensko zdravljenje s. lat. (15.1.)

Seminarji TehDNA

2013-10-08T13:39:33Z

EvaKnapič:

Seminarje iz Tehnologije DNA bo v študijskem letu 2013/14 vodila asist. dr. Helena Čelešnik.

Seznam tem za seminarje:

# Mutageneza (16.10.)
# Izražanje na površini (23.10.)
# Dvohibridni sistemi (30.10.)
# Mikromrežne tehnologije (6.11.)
# GSO v agronomiji (13.11.)
# Transgenske živali (27.11.) Andrea Grof
# Izvorne celice (4.12.) Sara Primec
# DNA-diagnostika (11.12.) Tina Gregorič , Eva Knapič
# Forenzika, arheologija, sistematika (18.12.)
# Mikromreže, genomike (8.1.)
# Gensko zdravljenje s. lat. (15.1.)

Odkritje kratke lasnične RNA (shRNA)

2012-04-02T20:22:30Z

EvaKnapič: /* Raziskave, ki so privedle do ustvarjanja trajnih celičnih linij sesalcev s pomočjo shRNA */

== Zgodovina pred poskusi z uporabo shRNA ==

Lin-4 in let-7 sta molekuli miRNA. Lin-4 so odkrili že davnega 1993. Ugotovili so, da je dolg okoli 20 nukleotidov in da nastane iz prekurzorske molekule, ki vsebuje lasnično zanko. Z različnimi poskusi so dokazali, da je lin-4 ključen za postembrionalni razvoj in da negativno regulira gene za določene proteine (gen lin-14 in lin-28). Mehanizem takrat še ni bil znan. Nekaj let kasneje so odkrili let-7 RNA, ki je približno iste velikosti kot lin-4 in prekurzor prav tako vsebuje lasnično zanko. Let-7 prav tako negativno regulira nekatere gene (lin-14...). Podobno kot lin-4, tudi let-7 sodeluje pri postembrionalnem razvoju (Lee et al. 1993, Pasquinelli et al. 2000, Relahart et al. 2000). Mehanizem je takrat prav tako bil neznan.

Čez par let so odkrili RNAi, njen mehanizem preko delovanja proteinov Dicer in RISC, tarčnega ciljanja mRNA in pojasnili, da sta lin-4 in let-7 miRNA. Kmalu zatem so znanstveniki želeli z uporabo umetne RNA inducirati inhibicijo določenih genov. Sprva so to poskušali z dsRNA. Prvi eksperimenti so potekali na C. Elegans. (Grishok et al. 2001, Hutvanger et al. 2001, Ketting et al. 2001). Pri poskusu so v črve injecirali dsRNA in opazovali odgovor. Opazili so, da se dsRNA spremeni v siRNA dolg približno 22nt. Si RNA nato sproži uničenje mRNA. Ključni pri omenjenem poskusu sta bila lin-4 in let-7. Znanstveniki so sklepali, da je prekurzor lin-4 in let-7 analogen dsRNA in miRNA istih prekurzorejv analogen siRNA. Za to obstaja več dokazov. Na kratko, za oba precesa so potrebni isti proteini (DCER-1, alg 1 in 2, red-1). Kmalu so podoben poskus izvedli na sesalcih, a je bil zaradi antivirusnega poskusa negativen. Pri sesalcih se zaradi dsRNA, posledično interferona, aktivira protein kinaza PKR. PKR nato sproži protivirusni odgovor in vodi celico v apoptozo (Gil in Esteban 2000). Tudi če v somatskih celicah zavremo PKR odziv, dsRNA sproži nespecifični odziv genske represije (Abraham et al., 1999; Paddison et al. 2001). Znanstveniki so zato skušali najti drugo pot, ki bi temeljila na istem principu. Ena izmed teh je bila konstrukcija shRNA z značilno lasnično zanko. Ta naj bi mimificirala prekurzorje miRNA, ki tudi vsebujejo lasnično zanko. Sh RNA tako v celici sproži RNAi odgovor. Do prvih uporab shRNA pri znanstvenih poskusih je prišlo leta 2002. Paddison et al., Brummelkamp et al., Paul et al., Sui et al.,Yu et al., Zeng et al., so ločeno injicirali spremenjeno dsRNA, ki je spominjala na endogeno shRNA in ni sprožila antivirusnega odgovora.

== Raziskave, ki so privedle do ustvarjanja trajnih celičnih linij sesalcev s pomočjo shRNA ==

Raziskovalci so želeli testirati možnost, da bi s posnemanjem lasnične RNA uspeli zmanipulirati gene tako, da bi določeni ostali utišani pri podvojevanju celic in pri dedovanju.

=== shRNA sproži utišanje genov v celicah ''Drosophila'' ===

Učinkovitost različnih kratkih lasničnih RNA, tako tistih s popolnim in nepopolnim ujemanjem s ciljnim substratom, so testirali na celicah ''Drosophila'' v S2 fazi.
Za najbolj učinkovite so se izkazale tiste z enostavno lasnično strukturo ter s popolno homologijo do substrata. Na učinkovitost ni vplivala velikost zanke ali sekvenca na zanki.
Rezultati so pokazali, da Dicer prepozna in predela shRNA, ki posnema njegov naravni substrat(let-7) in tudi tiste s preprosto lasnično strukturo.
Zaključili so, da je lahko rekombinantna shRNA preko Dicer-ja procesirana v siRNA in s tem potrdili prvotno idejo, da kratka lasnična zanka sproži utišanje genov preko mehanizma RNAi.

=== Utišanje genov v sesalskih celicah ===

Celice sesalcev vsebujejo endogene sisteme kot na primer PKR-preko dsRNA aktivirana protein kinaza, ki ovirajo delovanje RNAi. Da se kinaza aktivira, je potreben dupleks daljši od 30 baznih parov. Kratka RNA, ki posnema produkte Dicer-ja lahko obide sistem varovanja in utiša izražanje genov.
Učinkovitost shRNA pri utišanju so spremljali pri embrionalnih celicah človeških ledvic in jih primerjali z učinkovitostjo utišanja s siRNA. Rezultati so pokazali, da je bila v povprečju učinkovitost siRNA večja(90-95%) kot pri shRNA(80-90%). Pokazalo se je tudi, da so enostavnejše lasnične RNA pri utišanju uspešnejše od tistih, ki posnemajo naravni substrat encima Dicer.
Primerjali so tudi uspešnost različno dolgih shRNA. Največja učinkovitost se je pokazala pri shRNA dolgih od 25-29bp, znatno zmanjšanje pa se je pokazalo pri dupleksih krajših od 22bp.
Delovanje shRNA so potrdili še na rakavih celicah človeka, epitelnih celicah opic in fibroblastih glodalcev.

=== Sinteza shRNA ===

==== Sinteza shRNA ''in vitro'' – uporaba T7 RNA polimeraze ====

Transkripcijske templete za sintezo shRNA in siRNA so pripravili s pomočjo DNA sinteze. Tako sintetizirane shRNA in siRNA so preizkušali tako na celicah ''Drosophila'' in embrionalnih celicah človeških ledvic. Obe RNA sta pokazali enako učinkovitost kot njuni kemijsko sintetizirani različici. Problem se je pojavil pri sprožitvi sinteze, saj T7 polimeraza za začetek potrebuje dva gvanozinska ostanka, ki ju je potrebno dodati, da se sinteza lahko začne, ta pa lahko vplivata na delovanje siRNA. Zato je bolj uporabna sinteza shRNA, ki jo potem Dicer preoblikuje v aktivno obliko siRNA.

==== Sinteza shRNA ''in vivo'' – uporaba RNA polimeraze III ====

Preizkusili so želeli možnost sinteze na bolj naraven način z uporabo RNA polimeraze III. Promotorji RNA imajo dobro definirana začetna in končna mesta sinteze ter naravno sintetizirajo različne majhne in stabilne RNA. Dobro preučena promotorja U6 snRNA in H1 RNA sta omogočila manipuliranje promotorja do sinteze shRNA.
Sinteze so se lotili tako, da so v zaporedje tik za U6 snRNA promotorjem vstavili mesto kloniranja in s tem pridobili vektor, ki izkorišča lasnično zanko za utišanje genov. V vektor so klonirali shRNA zaporedje (pridobljeno iz luciferaze). S transfekcijo so vektor skupaj s plazmidom za izražanje luciferaze kresničke in ''Renilla'' vstavili v embrionalne celice človeških ledvic. Utišanje genov je bilo v obeh primerih shRNA učinkovito.
Preizkus zmožnosti vektorsko kodirane shRNA , da trajno utiša izražanje gena, so ustvarili lasnico, ki je ciljala na miškin p53 gen. Transfekcija z ras je v kontrolni kulturi celic povzročila prenehanje rasti, medtem ko so se v celični kulturi z vstavljenim shRNA vektorjem celice razmnoževale naprej še nekaj tednov. Analiza kultur pokaže, da je shRNA vektor utišal izražanje gena za p53 tako v prvotnih celicah kot tudi v njihovih potomkah.

Rezultati nakazujejo na to, da se lahko s pomočjo shRNA ustvari trajne celične linije sesalcev pri katerih je želeni gen trajno utišan.

== Bi-funkcionalna shRNA ==

Gre za najnovejšo tehnologijo, ki so jo opisali šele leta 2009 in je še vedno v razvoju. Bi-funkcionalna shRNA je sintetično majhno RNA, s pomočjo katere bi lahko manipulirali in vplivali na delovanje shRNA v celici. Razvili so jo, da bi z njeno pomočjo pospeši in povečali učinkovitost utišanja genov. Njena glavna prednost je ta, da je sestavljena iz dveh shRNA molekul, ki se lahko posamič vežeta ena na t.i. cepitveno-odvisen in druga na cepitveno-neodvisen RISC. Celotna bi-funkcionalna shRNA sestoji iz dveh stebličastih shRNA (stem-loop), ki se zaključita z zanko. V celico vstavijo plazmidni vektor, . Vstavljeni sta v miR-30 Ena ima v celoti ujemajoči verigi, deluje kot navadna shRNA in se na enak način pripne na cepitveno-odvisen del RISCa. Druga ima na sredini dva neujemajoča se dela, locirana na 9.-12. bazni par, ki omogočata vezavo na cepitveno-neodvisen RICS. Ko se končna produkta obeh shRNA vežeta na mRNA, tako hkrati inducirata transkripcijo in poskrbita za degradacijo mRNA. poskrbi, da prepis ne poteka. S takšnim, dopolnjujočim se delovanjem, poteka utišanje genov hitreje in bistveno učinkovitejše.

[[http://ars.sciencedirect.com/content/image/1-s2.0-S0169409X09000969-gr5.jpg Bi-funkcionalna shRNA]]

== Primerjava siRNA in miRNA s shRNA ==

siRNA je krajša in ima to posebnost, da se lahko nalaga na RICS brez, da bi šla preko vmesnega Dicer encimskega kompleksa. Komplementarno mRNA lahko cepi tako v citoplazmi kot v jedru. Ker so se pojavljali problemi ob uporabi siRNA pri poskusih ''in vivo'', so razvili novo, shRNA, ki je večja in lahko zato utiša večje dele genov. Nastane lahko s pomočjo transkripcije iz dsDNA z lasničnim zavojem, ki jo v celico dodamo preko ekspresorskega vektorja. Ko se shRNA enkrat veže na RISC, se utišanje genov nadaljuje po enakem mehanizmu kot pri siRNA. ShRNA v celici lahko nastane s transkripcijo iz ekspresorskega vektorja, ki ga v celico moramo dodati, medtem ko lahko siRNA nastane samo iz prekurzorja, ki je lahko bodisi shRNA ali pa dsRNA. Na splošno naj bi bilo delovanje shRNA učinkovitejše, ker jo celica sama sintetizira v jedru, medtem ko so za siRNA ugotovili, da se je v dveh dneh po vbrizgavanju, manj kot 1 % vseh siRNA vključil v mehanizem celice. Vendar pa vsem tem trditvam ne moremo povsem zaupati, saj so pri različnih raziskavah dobili precej različne rezultate. Prednost siRNA je v tem, da že majhna koncentracija teh molekul lahko zavre zapis nekega gena, medtem ko pri shRNA za isti efekt potrebujemo več molekul. Sklepamo lahko, da imata obe svoje prednosti in slabost. Za razliko od teh dveh RNAs, je miRNA endogena in sodeluje tudi pri številnih drugih celičnih procesih. Njena posebnost je tudi ta, da v nasprotju z drugima dvema ni potrebno popolno ujemanje z mRNA. Utiša lahko že gen, s katerim imata le delno komplementarno verigo, zato lahko vpliva na bistveno večje število genov. Lahko pa celo vpliva na samo DNA in tako regulira izražanje genov že pred nastankom mRNA.

== Viri ==

1. Grishok ''et al.'': Genes and Mechanisms Related to RNA InterferenceRegulate Expression of the Small Temporal RNAs that Control C. elegans Developmental Timing, ''Cell'' 2001, št. 106, str. 23-24

2. Rosalind C. Lee ''et al.'': An Extensive Class of Small RNAs in Caenorhabditis elegans. ''Science'' 2001, št. 294

3. Hutvagner ''et al.'': A Cellular Function for the RNA-Interference Enzyme Dicer in the Maturation of the let-7 Small Temporal RNA. ''Science'' 2001, št. 293

4. Paddison ''et al.'': RNA interference: the new somatic cell genetics?. Cancer cell, 2002,št. 2

5. Paddison P.J. ''et al.'': Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. ''Genes and Development'', 2002, letn. 16, str. 948-58.

6. Donald D. Ron ''et al.'': siRNA vs. shRNA: similarities and differences, ''Advanced Drug Delivery Reviews'', 2009, št. 61, str. 746-759

7. Wang Z. ''et al.'': RNA Interference and Cancer Therapy, ''Expert reviews'', 2011, št. 28, str. 2983-2995

[[Category:SEM]] [[Category:BMB]]

Talk:Odkritje kratke lasnične RNA (shRNA)

2012-04-02T20:18:19Z

EvaKnapič:

Alja Zottel:
Zgodovina pred poskusi z uporabo shRNA

Eva Knapič:
Raziskave, ki so privedle do ustvarjanja trajnih celičnih linij sesalcev s pomočjo shRNA

Taja Karner:
Bi-funkcionalna shRNA, Primerjava siRNA in miRNA s shRNA

Talk:Odkritje kratke lasnične RNA (shRNA)

2012-04-02T20:17:25Z

EvaKnapič:

Odkritje kratke lasnične RNA (shRNA)

2012-04-02T20:16:16Z

EvaKnapič: /* Sinteza shRNA in vitro – uporaba T7 RNA polimeraza */

== Zgodovina pred poskusi z uporabo shRNA ==

Lin-4 in let-7 sta molekuli miRNA. Lin-4 so odkrili že davnega 1993. Ugotovili so, da je dolg okoli 20 nukleotidov in da nastane iz prekurzorske molekule, ki vsebuje lasnično zanko. Z različnimi poskusi so dokazali, da je lin-4 ključen za postembrionalni razvoj in da negativno regulira gene za določene proteine (gen lin-14 in lin-28). Mehanizem takrat še ni bil znan. Nekaj let kasneje so odkrili let-7 RNA, ki je približno iste velikosti kot lin-4 in prekurzor prav tako vsebuje lasnično zanko. Let-7 prav tako negativno regulira nekatere gene (lin-14...). Podobno kot lin-4, tudi let-7 sodeluje pri postembrionalnem razvoju (Lee et al. 1993, Pasquinelli et al. 2000, Relahart et al. 2000). Mehanizem je takrat prav tako bil neznan.

Čez par let so odkrili RNAi, njen mehanizem preko delovanja proteinov Dicer in RISC, tarčnega ciljanja mRNA in pojasnili, da sta lin-4 in let-7 miRNA. Kmalu zatem so znanstveniki želeli z uporabo umetne RNA inducirati inhibicijo določenih genov. Sprva so to poskušali z dsRNA. Prvi eksperimenti so potekali na C. Elegans. (Grishok et al. 2001, Hutvanger et al. 2001, Ketting et al. 2001). Pri poskusu so v črve injecirali dsRNA in opazovali odgovor. Opazili so, da se dsRNA spremeni v siRNA dolg približno 22nt. Si RNA nato sproži uničenje mRNA. Ključni pri omenjenem poskusu sta bila lin-4 in let-7. Znanstveniki so sklepali, da je prekurzor lin-4 in let-7 analogen dsRNA in miRNA istih prekurzorejv analogen siRNA. Za to obstaja več dokazov. Na kratko, za oba precesa so potrebni isti proteini (DCER-1, alg 1 in 2, red-1). Kmalu so podoben poskus izvedli na sesalcih, a je bil zaradi antivirusnega poskusa negativen. Pri sesalcih se zaradi dsRNA, posledično interferona, aktivira protein kinaza PKR. PKR nato sproži protivirusni odgovor in vodi celico v apoptozo (Gil in Esteban 2000). Tudi če v somatskih celicah zavremo PKR odziv, dsRNA sproži nespecifični odziv genske represije (Abraham et al., 1999; Paddison et al. 2001). Znanstveniki so zato skušali najti drugo pot, ki bi temeljila na istem principu. Ena izmed teh je bila konstrukcija shRNA z značilno lasnično zanko. Ta naj bi mimificirala prekurzorje miRNA, ki tudi vsebujejo lasnično zanko. Sh RNA tako v celici sproži RNAi odgovor. Do prvih uporab shRNA pri znanstvenih poskusih je prišlo leta 2002. Paddison et al., Brummelkamp et al., Paul et al., Sui et al.,Yu et al., Zeng et al., so ločeno injicirali spremenjeno dsRNA, ki je spominjala na endogeno shRNA in ni sprožila antivirusnega odgovora.

== Raziskave, ki so privedle do ustvarjanja trajnih celičnih linij sesalcev s pomočjo shRNA ==

Raziskovalci so želeli testirati možnost, da bi s posnemanjem lasnične RNA uspeli zmanipulirati gene tako, da bi določeni ostali utišani pri podvojevanju celic in pri dedovanju.

=== shRNA sproži utišanje genov v celicah ''Drosophila'' ===

Učinkovitost različnih kratkih lasničnih RNA, tako tistih s popolnim in nepopolnim ujemanjem s ciljnim substratom, so testirali na celicah ''Drosophila'' v S2 fazi.
Za najbolj učinkovite so se izkazale tiste z enostavno lasnično strukturo ter s popolno homologijo do substrata. Na učinkovitost ni vplivala velikost zanke ali sekvenca na zanki.
Rezultati so pokazali, da Dicer prepozna in predela shRNA, ki posnema njegov naravni substrat(let-7) in tudi tiste s preprosto lasnično strukturo.
Zaključili so, da je lahko rekombinantna shRNA preko Dicer-ja procesirana v siRNA in s tem potrdili prvotno idejo, da kratka lasnična zanka sproži utišanje genov preko mehanizma RNAi.

=== Utišanje genov v sesalskih celicah ===

Celice sesalcev vsebujejo endogene sisteme kot na primer PKR-preko dsRNA aktivirana protein kinaza, ki ovirajo delovanje RNAi. Da se kinaza aktivira, je potreben dupleks daljši od 30 baznih parov. Kratka RNA, ki posnema produkte Dicer-ja lahko obide sistem varovanja in utiša izražanje genov.
Učinkovitost shRNA pri utišanju so spremljali pri embrionalnih celicah človeških ledvic in jih primerjali z učinkovitostjo utišanja s siRNA. Rezultati so pokazali, da je bila v povprečju učinkovitost siRNA večja(90-95%) kot pri shRNA(80-90%). Pokazalo se je tudi, da so enostavnejše lasnične RNA pri utišanju uspešnejše od tistih, ki posnemajo naravni substrat encima Dicer.
Primerjali so tudi uspešnost različno dolgih shRNA. Največja učinkovitost se je pokazala pri shRNA dolgih od 25-29bp, znatno zmanjšanje pa se je pokazalo pri dupleksih krajših od 22bp.
Delovanje shRNA so potrdili še na rakavih celicah človeka, epitelnih celicah opic in fibroblastih glodalcev.

=== Sinteza shRNA ===

==== Sinteza shRNA ''in vitro'' – uporaba T7 RNA polimeraza ====

Transkripcijske templete za sintezo shRNA in siRNA so pripravili s pomočjo DNA sinteze. Tako sintetizirane shRNA in siRNA so preizkušali tako na celicah ''Drosophila'' in embrionalnih celicah človeških ledvic. Obe RNA sta pokazali enako učinkovitost kot njuni kemijsko sintetizirani različici. Problem se je pojavil pri sprožitvi sinteze, saj T7 polimeraza za začetek potrebuje dva gvanozinska ostanka, ki ju je potrebno dodati, da se sinteza lahko začne, ta pa lahko vplivata na delovanje siRNA. Zato je bolj uporabna sinteza shRNA, ki jo potem Dicer preoblikuje v aktivno obliko siRNA.

==== Sinteza shRNA ''in vivo'' – uporaba RNA polimeraze III ====

Preizkusili so želeli možnost sinteze na bolj naraven način z uporabo RNA polimeraze III. Promotorji RNA imajo dobro definirana začetna in končna mesta sinteze ter naravno sintetizirajo različne majhne in stabilne RNA. Dobro preučena promotorja U6 snRNA in H1 RNA sta omogočila manipuliranje promotorja do sinteze shRNA.
Sinteze so se lotili tako, da so v zaporedje tik za U6 snRNA promotorjem vstavili mesto kloniranja in s tem pridobili vektor, ki izkorišča lasnično zanko za utišanje genov. V vektor so klonirali shRNA zaporedje (pridobljeno iz luciferaze). S transfekcijo so vektor skupaj s plazmidom za izražanje luciferaze kresničke in ''Renilla'' vstavili v embrionalne celice človeških ledvic. Utišanje genov je bilo v obeh primerih shRNA učinkovito.
Preizkus zmožnosti vektorsko kodirane shRNA , da trajno utiša izražanje gena, so ustvarili lasnico, ki je ciljala na miškin p53 gen. Transfekcija z ras je v kontrolni kulturi celic povzročila prenehanje rasti, medtem ko so se v celični kulturi z vstavljenim shRNA vektorjem celice razmnoževale naprej še nekaj tednov. Analiza kultur pokaže, da je shRNA vektor utišal izražanje gena za p53 tako v prvotnih celicah kot tudi v njihovih potomkah.

Rezultati nakazujejo na to, da se lahko s pomočjo shRNA ustvari trajne celične linije sesalcev pri katerih je želeni gen trajno utišan.

== Bi-funkcionalna shRNA ==

Gre za najnovejšo tehnologijo, ki so jo opisali šele leta 2009 in je še vedno v razvoju. Bi-funkcionalna shRNA je sintetično majhno RNA, s pomočjo katere bi lahko manipulirali in vplivali na delovanje shRNA v celici. Razvili so jo, da bi z njeno pomočjo pospeši in povečali učinkovitost utišanja genov. Njena glavna prednost je ta, da je sestavljena iz dveh shRNA molekul, ki se lahko posamič vežeta ena na t.i. cepitveno-odvisen in druga na cepitveno-neodvisen RISC. Celotna bi-funkcionalna shRNA sestoji iz dveh stebličastih shRNA (stem-loop), ki se zaključita z zanko. V celico vstavijo plazmidni vektor, . Vstavljeni sta v miR-30 Ena ima v celoti ujemajoči verigi, deluje kot navadna shRNA in se na enak način pripne na cepitveno-odvisen del RISCa. Druga ima na sredini dva neujemajoča se dela, locirana na 9.-12. bazni par, ki omogočata vezavo na cepitveno-neodvisen RICS. Ko se končna produkta obeh shRNA vežeta na mRNA, tako hkrati inducirata transkripcijo in poskrbita za degradacijo mRNA. poskrbi, da prepis ne poteka. S takšnim, dopolnjujočim se delovanjem, poteka utišanje genov hitreje in bistveno učinkovitejše.

[[http://ars.sciencedirect.com/content/image/1-s2.0-S0169409X09000969-gr5.jpg]]

== Primerjava siRNA in miRNA s shRNA ==

siRNA je krajša in ima to posebnost, da se lahko nalaga na RICS brez, da bi šla preko vmesnega Dicer encimskega kompleksa. Komplementarno mRNA lahko cepi tako v citoplazmi kot v jedru. Ker so se pojavljali problemi ob uporabi siRNA pri poskusih ''in vivo'', so razvili novo, shRNA, ki je večja in lahko zato utiša večje dele genov. Nastane lahko s pomočjo transkripcije iz dsDNA z lasničnim zavojem, ki jo v celico dodamo preko ekspresorskega vektorja. Ko se shRNA enkrat veže na RISC, se utišanje genov nadaljuje po enakem mehanizmu kot pri siRNA. ShRNA v celici lahko nastane s transkripcijo iz ekspresorskega vektorja, ki ga v celico moramo dodati, medtem ko lahko siRNA nastane samo iz prekurzorja, ki je lahko bodisi shRNA ali pa dsRNA. Na splošno naj bi bilo delovanje shRNA učinkovitejše, ker jo celica sama sintetizira v jedru, medtem ko so za siRNA ugotovili, da se je v dveh dneh po vbrizgavanju, manj kot 1 % vseh siRNA vključil v mehanizem celice. Vendar pa vsem tem trditvam ne moremo povsem zaupati, saj so pri različnih raziskavah dobili precej različne rezultate. Prednost siRNA je v tem, da že majhna koncentracija teh molekul lahko zavre zapis nekega gena, medtem ko pri shRNA za isti efekt potrebujemo več molekul. Sklepamo lahko, da imata obe svoje prednosti in slabost. Za razliko od teh dveh RNAs, je miRNA endogena in sodeluje tudi pri številnih drugih celičnih procesih. Njena posebnost je tudi ta, da v nasprotju z drugima dvema ni potrebno popolno ujemanje z mRNA. Utiša lahko že gen, s katerim imata le delno komplementarno verigo, zato lahko vpliva na bistveno večje število genov. Lahko pa celo vpliva na samo DNA in tako regulira izražanje genov že pred nastankom mRNA.

== Viri ==

1. Grishok ''et al.'': Genes and Mechanisms Related to RNA InterferenceRegulate Expression of the Small Temporal RNAs that Control C. elegans Developmental Timing, ''Cell'' 2001, št. 106, str. 23-24

2. Rosalind C. Lee ''et al.'': An Extensive Class of Small RNAs in Caenorhabditis elegans. ''Science'' 2001, št. 294

3. Hutvagner ''et al.'': A Cellular Function for the RNA-Interference Enzyme Dicer in the Maturation of the let-7 Small Temporal RNA. ''Science'' 2001, št. 293

4. Paddison ''et al.'': RNA interference: the new somatic cell genetics?. Cancer cell, 2002,št. 2

5. Paddison P.J. ''et al.'': Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. ''Genes and Development'', 2002, letn. 16, str. 948-58.

6. Donald D. Ron ''et al.'': siRNA vs. shRNA: similarities and differences, ''Advanced Drug Delivery Reviews'', 2009, št. 61, 746-759

7. Wang Z. ''et al.'': RNA Interference and Cancer Therapy, ''Expert reviews'', 2011, št. 28, 2983-2995

[[Category:SEM]] [[Category:BMB]]

Odkritje kratke lasnične RNA (shRNA)

2012-04-02T20:15:14Z

EvaKnapič: /* shRNA sproži utišanje genov v celicah Drosophila */

== Zgodovina pred poskusi z uporabo shRNA ==

Lin-4 in let-7 sta molekuli miRNA. Lin-4 so odkrili že davnega 1993. Ugotovili so, da je dolg okoli 20 nukleotidov in da nastane iz prekurzorske molekule, ki vsebuje lasnično zanko. Z različnimi poskusi so dokazali, da je lin-4 ključen za postembrionalni razvoj in da negativno regulira gene za določene proteine (gen lin-14 in lin-28). Mehanizem takrat še ni bil znan. Nekaj let kasneje so odkrili let-7 RNA, ki je približno iste velikosti kot lin-4 in prekurzor prav tako vsebuje lasnično zanko. Let-7 prav tako negativno regulira nekatere gene (lin-14...). Podobno kot lin-4, tudi let-7 sodeluje pri postembrionalnem razvoju (Lee et al. 1993, Pasquinelli et al. 2000, Relahart et al. 2000). Mehanizem je takrat prav tako bil neznan.

Čez par let so odkrili RNAi, njen mehanizem preko delovanja proteinov Dicer in RISC, tarčnega ciljanja mRNA in pojasnili, da sta lin-4 in let-7 miRNA. Kmalu zatem so znanstveniki želeli z uporabo umetne RNA inducirati inhibicijo določenih genov. Sprva so to poskušali z dsRNA. Prvi eksperimenti so potekali na C. Elegans. (Grishok et al. 2001, Hutvanger et al. 2001, Ketting et al. 2001). Pri poskusu so v črve injecirali dsRNA in opazovali odgovor. Opazili so, da se dsRNA spremeni v siRNA dolg približno 22nt. Si RNA nato sproži uničenje mRNA. Ključni pri omenjenem poskusu sta bila lin-4 in let-7. Znanstveniki so sklepali, da je prekurzor lin-4 in let-7 analogen dsRNA in miRNA istih prekurzorejv analogen siRNA. Za to obstaja več dokazov. Na kratko, za oba precesa so potrebni isti proteini (DCER-1, alg 1 in 2, red-1). Kmalu so podoben poskus izvedli na sesalcih, a je bil zaradi antivirusnega poskusa negativen. Pri sesalcih se zaradi dsRNA, posledično interferona, aktivira protein kinaza PKR. PKR nato sproži protivirusni odgovor in vodi celico v apoptozo (Gil in Esteban 2000). Tudi če v somatskih celicah zavremo PKR odziv, dsRNA sproži nespecifični odziv genske represije (Abraham et al., 1999; Paddison et al. 2001). Znanstveniki so zato skušali najti drugo pot, ki bi temeljila na istem principu. Ena izmed teh je bila konstrukcija shRNA z značilno lasnično zanko. Ta naj bi mimificirala prekurzorje miRNA, ki tudi vsebujejo lasnično zanko. Sh RNA tako v celici sproži RNAi odgovor. Do prvih uporab shRNA pri znanstvenih poskusih je prišlo leta 2002. Paddison et al., Brummelkamp et al., Paul et al., Sui et al.,Yu et al., Zeng et al., so ločeno injicirali spremenjeno dsRNA, ki je spominjala na endogeno shRNA in ni sprožila antivirusnega odgovora.

== Raziskave, ki so privedle do ustvarjanja trajnih celičnih linij sesalcev s pomočjo shRNA ==

Raziskovalci so želeli testirati možnost, da bi s posnemanjem lasnične RNA uspeli zmanipulirati gene tako, da bi določeni ostali utišani pri podvojevanju celic in pri dedovanju.

=== shRNA sproži utišanje genov v celicah ''Drosophila'' ===

Učinkovitost različnih kratkih lasničnih RNA, tako tistih s popolnim in nepopolnim ujemanjem s ciljnim substratom, so testirali na celicah ''Drosophila'' v S2 fazi.
Za najbolj učinkovite so se izkazale tiste z enostavno lasnično strukturo ter s popolno homologijo do substrata. Na učinkovitost ni vplivala velikost zanke ali sekvenca na zanki.
Rezultati so pokazali, da Dicer prepozna in predela shRNA, ki posnema njegov naravni substrat(let-7) in tudi tiste s preprosto lasnično strukturo.
Zaključili so, da je lahko rekombinantna shRNA preko Dicer-ja procesirana v siRNA in s tem potrdili prvotno idejo, da kratka lasnična zanka sproži utišanje genov preko mehanizma RNAi.

=== Utišanje genov v sesalskih celicah ===

Celice sesalcev vsebujejo endogene sisteme kot na primer PKR-preko dsRNA aktivirana protein kinaza, ki ovirajo delovanje RNAi. Da se kinaza aktivira, je potreben dupleks daljši od 30 baznih parov. Kratka RNA, ki posnema produkte Dicer-ja lahko obide sistem varovanja in utiša izražanje genov.
Učinkovitost shRNA pri utišanju so spremljali pri embrionalnih celicah človeških ledvic in jih primerjali z učinkovitostjo utišanja s siRNA. Rezultati so pokazali, da je bila v povprečju učinkovitost siRNA večja(90-95%) kot pri shRNA(80-90%). Pokazalo se je tudi, da so enostavnejše lasnične RNA pri utišanju uspešnejše od tistih, ki posnemajo naravni substrat encima Dicer.
Primerjali so tudi uspešnost različno dolgih shRNA. Največja učinkovitost se je pokazala pri shRNA dolgih od 25-29bp, znatno zmanjšanje pa se je pokazalo pri dupleksih krajših od 22bp.
Delovanje shRNA so potrdili še na rakavih celicah človeka, epitelnih celicah opic in fibroblastih glodalcev.

=== Sinteza shRNA ===

==== Sinteza shRNA ''in vitro'' – uporaba T7 RNA polimeraza ====

Transkripcijske templete za sintezo shRNA in siRNA so pripravili s pomočjo DNA sinteze. Tako sintetizirane shRNA in siRNA so preizkušali tako na celicah Drosophila in embrionalnih celicah človeških ledvic. Obe RNA sta pokazali enako učinkovitost kot njuni kemijsko sintetizirani različici. Problem se je pojavil pri sprožitvi sinteze, saj T7 polimeraza za začetek potrebuje dva gvanozinska ostanka, ki ju je potrebno dodati, da se sinteza lahko začne, ta pa lahko vplivata na delovanje siRNA. Zato je bolj uporabna sinteza shRNA, ki jo potem Dicer preoblikuje v aktivno obliko siRNA.

==== Sinteza shRNA ''in vivo'' – uporaba RNA polimeraze III ====

Preizkusili so želeli možnost sinteze na bolj naraven način z uporabo RNA polimeraze III. Promotorji RNA imajo dobro definirana začetna in končna mesta sinteze ter naravno sintetizirajo različne majhne in stabilne RNA. Dobro preučena promotorja U6 snRNA in H1 RNA sta omogočila manipuliranje promotorja do sinteze shRNA.
Sinteze so se lotili tako, da so v zaporedje tik za U6 snRNA promotorjem vstavili mesto kloniranja in s tem pridobili vektor, ki izkorišča lasnično zanko za utišanje genov. V vektor so klonirali shRNA zaporedje (pridobljeno iz luciferaze). S transfekcijo so vektor skupaj s plazmidom za izražanje luciferaze kresničke in ''Renilla'' vstavili v embrionalne celice človeških ledvic. Utišanje genov je bilo v obeh primerih shRNA učinkovito.
Preizkus zmožnosti vektorsko kodirane shRNA , da trajno utiša izražanje gena, so ustvarili lasnico, ki je ciljala na miškin p53 gen. Transfekcija z ras je v kontrolni kulturi celic povzročila prenehanje rasti, medtem ko so se v celični kulturi z vstavljenim shRNA vektorjem celice razmnoževale naprej še nekaj tednov. Analiza kultur pokaže, da je shRNA vektor utišal izražanje gena za p53 tako v prvotnih celicah kot tudi v njihovih potomkah.

Rezultati nakazujejo na to, da se lahko s pomočjo shRNA ustvari trajne celične linije sesalcev pri katerih je želeni gen trajno utišan.

== Bi-funkcionalna shRNA ==

Gre za najnovejšo tehnologijo, ki so jo opisali šele leta 2009 in je še vedno v razvoju. Bi-funkcionalna shRNA je sintetično majhno RNA, s pomočjo katere bi lahko manipulirali in vplivali na delovanje shRNA v celici. Razvili so jo, da bi z njeno pomočjo pospeši in povečali učinkovitost utišanja genov. Njena glavna prednost je ta, da je sestavljena iz dveh shRNA molekul, ki se lahko posamič vežeta ena na t.i. cepitveno-odvisen in druga na cepitveno-neodvisen RISC. Celotna bi-funkcionalna shRNA sestoji iz dveh stebličastih shRNA (stem-loop), ki se zaključita z zanko. V celico vstavijo plazmidni vektor, . Vstavljeni sta v miR-30 Ena ima v celoti ujemajoči verigi, deluje kot navadna shRNA in se na enak način pripne na cepitveno-odvisen del RISCa. Druga ima na sredini dva neujemajoča se dela, locirana na 9.-12. bazni par, ki omogočata vezavo na cepitveno-neodvisen RICS. Ko se končna produkta obeh shRNA vežeta na mRNA, tako hkrati inducirata transkripcijo in poskrbita za degradacijo mRNA. poskrbi, da prepis ne poteka. S takšnim, dopolnjujočim se delovanjem, poteka utišanje genov hitreje in bistveno učinkovitejše.

[[http://ars.sciencedirect.com/content/image/1-s2.0-S0169409X09000969-gr5.jpg]]

== Primerjava siRNA in miRNA s shRNA ==

siRNA je krajša in ima to posebnost, da se lahko nalaga na RICS brez, da bi šla preko vmesnega Dicer encimskega kompleksa. Komplementarno mRNA lahko cepi tako v citoplazmi kot v jedru. Ker so se pojavljali problemi ob uporabi siRNA pri poskusih ''in vivo'', so razvili novo, shRNA, ki je večja in lahko zato utiša večje dele genov. Nastane lahko s pomočjo transkripcije iz dsDNA z lasničnim zavojem, ki jo v celico dodamo preko ekspresorskega vektorja. Ko se shRNA enkrat veže na RISC, se utišanje genov nadaljuje po enakem mehanizmu kot pri siRNA. ShRNA v celici lahko nastane s transkripcijo iz ekspresorskega vektorja, ki ga v celico moramo dodati, medtem ko lahko siRNA nastane samo iz prekurzorja, ki je lahko bodisi shRNA ali pa dsRNA. Na splošno naj bi bilo delovanje shRNA učinkovitejše, ker jo celica sama sintetizira v jedru, medtem ko so za siRNA ugotovili, da se je v dveh dneh po vbrizgavanju, manj kot 1 % vseh siRNA vključil v mehanizem celice. Vendar pa vsem tem trditvam ne moremo povsem zaupati, saj so pri različnih raziskavah dobili precej različne rezultate. Prednost siRNA je v tem, da že majhna koncentracija teh molekul lahko zavre zapis nekega gena, medtem ko pri shRNA za isti efekt potrebujemo več molekul. Sklepamo lahko, da imata obe svoje prednosti in slabost. Za razliko od teh dveh RNAs, je miRNA endogena in sodeluje tudi pri številnih drugih celičnih procesih. Njena posebnost je tudi ta, da v nasprotju z drugima dvema ni potrebno popolno ujemanje z mRNA. Utiša lahko že gen, s katerim imata le delno komplementarno verigo, zato lahko vpliva na bistveno večje število genov. Lahko pa celo vpliva na samo DNA in tako regulira izražanje genov že pred nastankom mRNA.

== Viri ==

1. Grishok ''et al.'': Genes and Mechanisms Related to RNA InterferenceRegulate Expression of the Small Temporal RNAs that Control C. elegans Developmental Timing, ''Cell'' 2001, št. 106, str. 23-24

2. Rosalind C. Lee ''et al.'': An Extensive Class of Small RNAs in Caenorhabditis elegans. ''Science'' 2001, št. 294

3. Hutvagner ''et al.'': A Cellular Function for the RNA-Interference Enzyme Dicer in the Maturation of the let-7 Small Temporal RNA. ''Science'' 2001, št. 293

4. Paddison ''et al.'': RNA interference: the new somatic cell genetics?. Cancer cell, 2002,št. 2

5. Paddison P.J. ''et al.'': Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. ''Genes and Development'', 2002, letn. 16, str. 948-58.

6. Donald D. Ron ''et al.'': siRNA vs. shRNA: similarities and differences, ''Advanced Drug Delivery Reviews'', 2009, št. 61, 746-759

7. Wang Z. ''et al.'': RNA Interference and Cancer Therapy, ''Expert reviews'', 2011, št. 28, 2983-2995

[[Category:SEM]] [[Category:BMB]]

BIO2 Seminar 2011

2011-11-18T13:53:13Z

EvaKnapič: /* Seznam seminarjev- datumi in seznam recenzentov še niso dokončni! */

= Biokemijski seminar =

Seminarje vodi doc. dr. Gregor Gunčar in so na urniku vsako sredo in petek po eni uri predavanj iz Biokemije.

Ocena seminarjev predstavlja 30% končne ocene in vsebuje vse točke, ki jih študent/ka lahko zbere pri seminarju in ostalih dejavnostih, ki niso del pisnega izpita.

== Seznam seminarjev- datumi in seznam recenzentov še niso dokončni! ==
Vpišite svoj izbrani naslov!!!
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Ime in priimek'''
| align="center" style="background:#f0f0f0;"|'''Naslov seminarja'''
| align="center" style="background:#f0f0f0;"|'''Rok za oddajo'''
| align="center" style="background:#f0f0f0;"|'''Rok za recenzijo'''
| align="center" style="background:#f0f0f0;"|'''Datum predstavitve'''
| align="center" style="background:#f0f0f0;"|'''Recenzent1'''
| align="center" style="background:#f0f0f0;"|'''Recenzent2'''
|-
| Ula Štok||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011 Tipping the mind]||17.10.11||19.10.11||21.10.11||Maja Remškar||Mirjam Kmetič
|-
| Maša Mirković||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011 The twisted way of things]||17.10.11||19.10.11||21.10.11||Eva Knapič||Marko Radojković
|-
| Sara Draščič||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011 On the spur of a whim ]||17.10.11||19.10.11||21.10.11||Matevž Merljak||Monika Škrjanc
|-
| Katra Koman||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Katra_Koman:_INZULIN Protein of the 20th century]||18.10.11||23.10.11||26.10.11||Ines Kerin||Veronika Jarc
|-
| Ana Dolinar||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Ana_Dolinar:_Univerzalna_kri_.E2.80.93_prihodnost_transfuzijske_medicine.3F The juice of life]||21.10.11||25.10.11||28.10.11||Tjaša Goričan||Andreja Bratovš
|-
| Urška Rauter||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Ur.C5.A1ka_Rauter:_A_Green_Glow:_zgradba_in_funkcija_encima_luciferaze A green glow]||21.10.11||25.10.11||28.10.11||Maša Mohar||Sandi Botonjić
|-
| Taja Karner||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Taja_Karner:_Glavoboli_in_migrene Throb]||21.10.11||26.10.11||02.11.11||Karmen Hrovat||Tamara Marić
|-
| Rok Štemberger||Forbidden fruit||21.10.11||28.10.11||04.11.11||Špela Pohleven||Maja Grdadolnik
|-
| Maša Mohar||The tenuous nature of sex||21.10.11||28.10.11||04.11.11||Andreja Bratovš||Ines Kerin
|-
| Veronika Jarc||Our hollow architecture||21.10.11||28.10.11||04.11.11||Sabina Mavretič||Matevž Ambrožič
|-
| Mirjam Kmetič||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Mirjam_Kmeti.C4.8D:_Mint_condition_.28limonen-3-hidroksilaza_in_limonen-6-hidroksilaza.29 Mint condition]||26.10.11||02.11.11||09.11.11||Sandi Botonjić||Tina Gregorič
|-
| Janez Meden||The Japanese Horseshoe Crab and Deafness||28.10.11||01.12.11||20.1.12||Veronika Jarc||Ana Dolinar
|-
| Tjaša Flis||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Sandi_Botonji.C4.87:_Kokain_esteraza Life's tremors]||28.10.11||04.11.11||11.11.11||Ana Dolinar||Špela Pohleven
|-
| Sandi Botonjić||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Sandi_Botonji.C4.87:_Kokain_esteraza Nature's junkie]||28.10.11||04.11.11||11.11.11||Maša Mirković||Alenka Mikuž
|-
| Kaja Javoršek||A grey matter||02.11.11||09.11.11||16.11.11||Dominik Kert||Tjaša Flis
|-
| Rok Vene||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Rok_Vene:_A_mind_astray A mind astray]||04.11.11||11.11.11||18.11.11||Tamara Marić||Maja Remškar
|-
| Ines Šterbal||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011 LTP1]||04.11.11||11.11.11||18.11.11||Ula Štok||Rok Vene
|-
| Matja Zalar||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Matja_Zalar:_Vloga_SRK_in_SCR_proteinov_pri_prepre.C4.8Devanju_incestnega_razmno.C5.BEevanja_c Do it yourself]||04.11.11||11.11.11||18.11.11||Monika Škrjanc||Matevž Merljak
|-
| Matevž Ambrožič||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Matev.C5.BE_Ambro.C5.BEi.C4.8D:_BSX_protein_in_debelost Of fidgets and food]||09.11.11||16.11.11||23.11.11||Kaja Javoršek||Petra Malavašič
|-
| Matevž Merljak||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Matev.C5.BE_Merljak:_CEM15.2C_VIF_in_infektivnost_retrovirusov Protein wars]||11.11.11||18.11.11||25.11.11||Teja Banič||Urška Navodnik
|-
| Mitja Crček||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Mijta_Cr.C4.8Dek:_DSIP_in_spanje When your day draws to an end]||11.11.11||18.11.11||25.11.11||Marko Radojković||Andrej Vrankar
|-
| Dominik Kert||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Dominik_Kert:_FOXP2.2C_govore.C4.8Di_protein FOXP2, govoreči protein]||11.11.11||18.11.11||25.11.11||Alja Zottel||Kaja Javoršek
|-
| Petra Malavašič||Going unnoticed||16.11.11||23.11.11||30.11.11||Maja Grdadolnik||Mitja Crček
|-
| Eva Knapič||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Eva_Knapi.C4.8D:_TSH3_-_Kaj_novorojen.C4.8Dkom_omogo.C4.8Da_zadihati? Life's first breath]||18.11.11||25.11.11||02.12.11||Mirjam Kmetič||Andrej Vrankar
|-
| Marko Radojković||Paint my thoughts||18.11.11||25.11.11||02.12.11||Sara Draščič||Urška Rode
|-
| Tjaša Goričan||Nerve regrowth: nipped by a no-go||18.11.11||25.11.11||02.12.11||Ana Remžgar||Ines Šterbal
|-
| Tina Gregorič||Gut feelings||23.11.11||30.11.11||07.12.11||Janez Meden||Urška Rauter
|-
| Tamara Marić||The dark side of RNA||25.11.11||02.12.11||09.12.11||Dominik Kert||Rok Štemberger
|-
| Ana Remžgar||I'll have you for supper||25.11.11||02.12.11||09.12.11||Jana Verbančič||Eva Knapič
|-
| Maja Remškar||Questioning Colour||25.11.11||02.12.11||09.12.11||Katra Koman||Karmen Belšak
|-
| Andreja Bratovš||The power behind pain||30.11.11||07.12.11||14.12.11||Matevž Ambrožič||Teja Banič
|-
| Urška Navodnik||Darwin\'s dessert||02.12.11||09.12.11||16.12.11||Taja Karner||Karmen Hrovat
|-
| Jernej Mustar||Silent pain||02.12.11||09.12.11||16.12.11||Petra Malavašič||Jana Verbančič
|-
| Ines Kerin||A queen\'s dinner||02.12.11||09.12.11||16.12.11||Tjaša Flis||Iza Ogris
|-
| Alja Zottel||Sleepless nights||07.12.11||14.12.11||21.12.11||Ines Šterbal||Katra Koman
|-
| Alenka Mikuž||Molecular chastity||09.12.11||16.12.11||23.12.11||Urška Rode||Janez Meden
|-
| Maja Grdadolnik||Ear of Stone||09.12.11||16.12.11||23.12.11||Tina Gregorič||Ana Potočnik
|-
| Jana Verbančič||A balanced mind||09.12.11||16.12.11||23.12.11||Alenka Mikuž||Ana Remžgar
|-
| Karmen Hrovat||The thread of life||14.12.11||21.12.11||04.01.12||Iza Ogris||Taja Karner
|-
| Andrej Vrankar||The things we forget||16.12.11||23.12.11||06.01.12||Jernej Mustar||Maša Mohar
|-
| Teja Banič||Cool news||16.12.11||23.12.11||06.01.12||Karmen Belšak||Jernej Mustar
|-
| Špela Pohleven||The making of crooked||16.12.11||23.12.11||06.01.12||Mitja Crček||Maša Mirković
|-
| Sabina Mavretič||A short story||21.12.11||04.01.12||11.01.12||Rok Vene||Sabina Mavretič
|-
| Karmen Belšak||Another dark horse||23.12.11||06.01.12||13.01.12||Urška Rauter||Sara Draščič
|-
| Iza Ogris||Love,love, love...||23.12.11||06.01.12||13.01.12||Ana Potočnik||Matja Zalar
|-
| Monika Škrjanc||The greenest of us all||23.12.11||06.01.12||13.01.12||Rok Štemberger||Tjaša Goričan
|-
| Ana Potočnik||Skin-deep||04.01.12||11.01.12||18.01.12||Matja Zalar||Ula Štok
|-
| Urška Rode||Smart sweat||06.01.12||13.01.12||20.01.12||Urška Navodnik||Alja Zottel
|-
| Ime in priimek||Naslov seminarja||06.01.12||13.01.12||20.01.12||||
|}

== Gradivo za seminarje ==
NOVO Gradivo za predavanja in seminarje najdete na http://bio.ijs.si/~zajec/bio2/
username: bio2
password: samozame

==Naloga==
'''Vaša naloga za seminar je: '''
Samostojno pripraviti seminar o enem od proteinov opisanih v [http://web.expasy.org/spotlight/back_issues/2011/ ProteinSpotlight] Poiskati morate vsaj še tri znanstvene članke, ki se nanašajo na opisano temo in jih uporabiti kot podlago za seminarsko nalogo!

V okviru seminarske naloge morate opraviti še naslednje naloge, katerih rešitve predložite na dodatni strani seminarske naloge, ki se ne šteje v kvoto obsega seminarja:

* sekvenca proteina in [http://www.uniprot.org/ UniProt] oznaka proteina
* slika strukture proteina (če je le-ta znana), ki jo naredite sami s programom Pymol. Če struktura še ni znana, vključite sliko proteina, ki je vašemu najbolj podoben po sekvenci in katerega struktura je znana
* poiskati morate, na katerem kromosomu se v človeškem genu nahaja ta protein in narisati shematsko sliko gena (eksonov in intronov) tega proteina. Če protein ni človeškega izvora, poiščite protein, ki je vašemu najbolj podoben in vse navedeno opišite za ta protein.

Za pripravo seminarja velja naslednje: 
* [[BIO2 Povzetki seminarjev 2011|Povzetek seminarja]] opišete na wikiju v približno 200 besedah - najkasneje do dne ko morate oddati seminar recenzentom.
* Povezavo do povzetka vnesete v tabelo seminarjev tekočega letnika.
* Seminar pripravite v obliki seminarske naloge na ~5-9 straneh A4 (pisava 12, enojni razmak, 2,5 cm robovi; važno je, da je obseg od 2700 do 3000 besed), vsebovati mora najmanj tri slike. Slika mora imeti legendo in v besedilu mora biti na ustreznem mestu sklic na sliko.
* Seminar oddajte do datuma oddaje, ki je naveden v tabeli vsakemu od recenzentov in docentu (docentu ga pošljite po e-pošti).
* Recenzenti do dneva določenega v tabeli določijo popravke in podajo oceno pisnega dela.
* Ustna predstavitev sledi na dan, ki je vpisan v tabeli. Za predstavitev je na voljo 20-30 minut. Recenzenti morajo biti na predstavitvi prisotni.
* Predstavitvi sledi razprava. Recenzenti podajo oceno predstavitve in postavijo najmanj dve vprašanji.
* Na dan predstavitve morate docentu oddati končno (popravljeno) in natisnjeno verzijo seminarja v enem izvodu.
* Seminarska naloga in povzetek morajo biti v slovenskem jeziku!

==Ocenjevanje seminarjev==
Recenzenti ocenijo seminar tako, da izpolnijo [https://docs.google.com/spreadsheet/viewform?formkey=dG1Pa3p2NXE2Vm1zX3FpVTZCT2dHVnc6MA recenzentsko poročilo] na spletu.

== Mnenje o predstavitvi ==
Vsak posameznik '''mora''' oceniti seminar, tako da odda svoje [https://docs.google.com/spreadsheet/viewform?formkey=dFNXUDBCRVBaVExvOFVxakpJUHRnOEE6MA mnenje] najkasneje v šestih dneh po predstavitvi. Kdor na seminarju ni bil prisoten, mnenja '''ne sme''' oddati.

==Urejanje spletnih strani na wikiju==
Wiki so razvili zato, da lahko spletne vsebine ureja vsakdo. Ukazi so preprosti, dokler si ne zamislite česa prav posebnega. Vseeno pa je Word v primerjavi z wikijem pravo čudežno orodje... Če imate težave z oblikovanjem besedila, si preberite poglavje o urejanju wiki-strani na Wikipediji ([http://en.wikipedia.org/wiki/Help:Editing tule] v angleščini in [http://sl.wikipedia.org/wiki/Wikipedija:Urejanje_strani tu] v slovenščini). Pomaga tudi, če pogledate, kako je zapisana kakšna stran, ki se vam zdi v redu: kliknite na zavihek 'Uredite stran' in si poglejte, kako so vpisane povezave, kako nov odstavek in podobno. ''Na koncu seveda pod oknom za urejanje kliknite na 'Prekliči'.''

==Citiranje virov==
Citiranje je možno po več shemah, važno je, da se v seminarju držite ene same.
Temeljno načelo je, da je treba vir navesti na tak način, da ga je mogoče nedvoumno poiskati.
Za citate v naravoslovju je najpogostejše citiranje po pravilniku ISO 690. [http://www.zveza-zotks.si/gzm/dokumenti/literatura.html Pravila], ki upoštevajo omenjeni standard, so pripravili pri ZTKS. Sicer pa ima vsaka revija lahko svoj način citiranja, ki ga je treba pri pisanju članka upoštevati. 
'''Citiranje knjig:''' 
Priimek, I. ''Naslov''. Kraj: Založba, letnica. 
Priimek, I. ''Naslov: podnaslov''. Izdaja. Kraj: Založba, letnica. Zbirka, številka. ISBN. 

Boyer, R. ''Temelji biokemije''. Ljubljana: Študentska založba, 2005. 
Glick BR in Pasternak JJ. ''Molecular biotechnology: principles and applications of recombinant DNA''. 3. izdaja. Washington: ASM Press, 2003. ISBN 1-55581-269-4. 
Če so avtorji trije, je beseda in med drugim in tretjim avtorjem. Če so avtorji več kot trije, napišemo samo prvega in dopišemo ''et al''. (in drugi, po latinsko). Vse, kar je latinsko, pišemo poševno (npr. tudi imena rastlin in živali, pojme ''in vivo'', ''in vitro'' ipd.).

'''Citiranje člankov:''' 
Priimek, I. Naslov. ''Naslov revije'', letnica, letnik, številka, strani. 
Lartigue C. ''et al''. Genome transplantation in bacteria: changing one species to another. ''Science'', 2007, letn. 317, str. 632-638.

Alternativni način citiranja (predvsem v družboslovju) je po pravilih APA, kjer članke citirajo takole: 
Priimek, I. (letnica, številka). Naslov. Naslov revije, strani. 
Lartigue C. ''et al.'' (2007, 317) Genome transplantation in bacteria: changing one species to another. ''Science'', 632-638.

Revija Science uporablja skrajšani zapis: 
C. Lartigue ''et al''. Science 317, 632 (2007) 

V diplomah na FKKT je treba navesti vire tako, da izpišete tudi naslov citiranega dela in strani od-do (ne samo začetne).

'''Citiranje spletnih virov:''' 
Priimek, I. ''Naslov dokumenta''. Izdaja. Kraj: Založnik, letnica. Datum zadnjega popravljanja. [Datum citiranja.] spletni naslov 
strangeguitars. ''On the brink of artificial life''. 6. 10. 2007. [citirano 13. 11. 2007] http://www.metafilter.com/65331/On-the-brink-of-artificial-life 
Navedemo čim več podatkov; pogosto vseh iz pravila ne boste našli.

BIO2 Seminar 2011

2011-11-18T13:39:42Z

EvaKnapič: /* Seznam seminarjev- datumi in seznam recenzentov še niso dokončni! */

= Biokemijski seminar =

Seminarje vodi doc. dr. Gregor Gunčar in so na urniku vsako sredo in petek po eni uri predavanj iz Biokemije.

Ocena seminarjev predstavlja 30% končne ocene in vsebuje vse točke, ki jih študent/ka lahko zbere pri seminarju in ostalih dejavnostih, ki niso del pisnega izpita.

== Seznam seminarjev- datumi in seznam recenzentov še niso dokončni! ==
Vpišite svoj izbrani naslov!!!
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Ime in priimek'''
| align="center" style="background:#f0f0f0;"|'''Naslov seminarja'''
| align="center" style="background:#f0f0f0;"|'''Rok za oddajo'''
| align="center" style="background:#f0f0f0;"|'''Rok za recenzijo'''
| align="center" style="background:#f0f0f0;"|'''Datum predstavitve'''
| align="center" style="background:#f0f0f0;"|'''Recenzent1'''
| align="center" style="background:#f0f0f0;"|'''Recenzent2'''
|-
| Ula Štok||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011 Tipping the mind]||17.10.11||19.10.11||21.10.11||Maja Remškar||Mirjam Kmetič
|-
| Maša Mirković||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011 The twisted way of things]||17.10.11||19.10.11||21.10.11||Eva Knapič||Marko Radojković
|-
| Sara Draščič||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011 On the spur of a whim ]||17.10.11||19.10.11||21.10.11||Matevž Merljak||Monika Škrjanc
|-
| Katra Koman||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Katra_Koman:_INZULIN Protein of the 20th century]||18.10.11||23.10.11||26.10.11||Ines Kerin||Veronika Jarc
|-
| Ana Dolinar||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Ana_Dolinar:_Univerzalna_kri_.E2.80.93_prihodnost_transfuzijske_medicine.3F The juice of life]||21.10.11||25.10.11||28.10.11||Tjaša Goričan||Andreja Bratovš
|-
| Urška Rauter||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Ur.C5.A1ka_Rauter:_A_Green_Glow:_zgradba_in_funkcija_encima_luciferaze A green glow]||21.10.11||25.10.11||28.10.11||Maša Mohar||Sandi Botonjić
|-
| Taja Karner||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Taja_Karner:_Glavoboli_in_migrene Throb]||21.10.11||26.10.11||02.11.11||Karmen Hrovat||Tamara Marić
|-
| Rok Štemberger||Forbidden fruit||21.10.11||28.10.11||04.11.11||Špela Pohleven||Maja Grdadolnik
|-
| Maša Mohar||The tenuous nature of sex||21.10.11||28.10.11||04.11.11||Andreja Bratovš||Ines Kerin
|-
| Veronika Jarc||Our hollow architecture||21.10.11||28.10.11||04.11.11||Sabina Mavretič||Matevž Ambrožič
|-
| Mirjam Kmetič||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Mirjam_Kmeti.C4.8D:_Mint_condition_.28limonen-3-hidroksilaza_in_limonen-6-hidroksilaza.29 Mint condition]||26.10.11||02.11.11||09.11.11||Sandi Botonjić||Tina Gregorič
|-
| Janez Meden||The Japanese Horseshoe Crab and Deafness||28.10.11||01.12.11||20.1.12||Veronika Jarc||Ana Dolinar
|-
| Tjaša Flis||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Sandi_Botonji.C4.87:_Kokain_esteraza Life's tremors]||28.10.11||04.11.11||11.11.11||Ana Dolinar||Špela Pohleven
|-
| Sandi Botonjić||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Sandi_Botonji.C4.87:_Kokain_esteraza Nature's junkie]||28.10.11||04.11.11||11.11.11||Maša Mirković||Alenka Mikuž
|-
| Kaja Javoršek||A grey matter||02.11.11||09.11.11||16.11.11||Dominik Kert||Tjaša Flis
|-
| Rok Vene||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Rok_Vene:_A_mind_astray A mind astray]||04.11.11||11.11.11||18.11.11||Tamara Marić||Maja Remškar
|-
| Ines Šterbal||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011 LTP1]||04.11.11||11.11.11||18.11.11||Ula Štok||Rok Vene
|-
| Matja Zalar||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Matja_Zalar:_Vloga_SRK_in_SCR_proteinov_pri_prepre.C4.8Devanju_incestnega_razmno.C5.BEevanja_c Do it yourself]||04.11.11||11.11.11||18.11.11||Monika Škrjanc||Matevž Merljak
|-
| Matevž Ambrožič||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Matev.C5.BE_Ambro.C5.BEi.C4.8D:_BSX_protein_in_debelost Of fidgets and food]||09.11.11||16.11.11||23.11.11||Kaja Javoršek||Petra Malavašič
|-
| Matevž Merljak||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Matev.C5.BE_Merljak:_CEM15.2C_VIF_in_infektivnost_retrovirusov Protein wars]||11.11.11||18.11.11||25.11.11||Teja Banič||Urška Navodnik
|-
| Mitja Crček||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Mijta_Cr.C4.8Dek:_DSIP_in_spanje When your day draws to an end]||11.11.11||18.11.11||25.11.11||Marko Radojković||Andrej Vrankar
|-
| Dominik Kert||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Dominik_Kert:_FOXP2.2C_govore.C4.8Di_protein FOXP2, govoreči protein]||11.11.11||18.11.11||25.11.11||Alja Zottel||Kaja Javoršek
|-
| Petra Malavašič||Going unnoticed||16.11.11||23.11.11||30.11.11||Maja Grdadolnik||Mitja Crček
|-
| Eva Knapič||[http://wiki.fkkt.uni-lj.si/index.php/BIO2_Povzetki_seminarjev_2011#Eva_Knapič:_TSH3_-_Kaj_novorojenčkov_omogoča_zadihati Life's first breath]||18.11.11||25.11.11||02.12.11||Mirjam Kmetič||Andrej Vrankar
|-
| Marko Radojković||Paint my thoughts||18.11.11||25.11.11||02.12.11||Sara Draščič||Urška Rode
|-
| Tjaša Goričan||Nerve regrowth: nipped by a no-go||18.11.11||25.11.11||02.12.11||Ana Remžgar||Ines Šterbal
|-
| Tina Gregorič||Gut feelings||23.11.11||30.11.11||07.12.11||Janez Meden||Urška Rauter
|-
| Tamara Marić||The dark side of RNA||25.11.11||02.12.11||09.12.11||Dominik Kert||Rok Štemberger
|-
| Ana Remžgar||I'll have you for supper||25.11.11||02.12.11||09.12.11||Jana Verbančič||Eva Knapič
|-
| Maja Remškar||Questioning Colour||25.11.11||02.12.11||09.12.11||Katra Koman||Karmen Belšak
|-
| Andreja Bratovš||The power behind pain||30.11.11||07.12.11||14.12.11||Matevž Ambrožič||Teja Banič
|-
| Urška Navodnik||Darwin\'s dessert||02.12.11||09.12.11||16.12.11||Taja Karner||Karmen Hrovat
|-
| Jernej Mustar||Silent pain||02.12.11||09.12.11||16.12.11||Petra Malavašič||Jana Verbančič
|-
| Ines Kerin||A queen\'s dinner||02.12.11||09.12.11||16.12.11||Tjaša Flis||Iza Ogris
|-
| Alja Zottel||Sleepless nights||07.12.11||14.12.11||21.12.11||Ines Šterbal||Katra Koman
|-
| Alenka Mikuž||Molecular chastity||09.12.11||16.12.11||23.12.11||Urška Rode||Janez Meden
|-
| Maja Grdadolnik||Ear of Stone||09.12.11||16.12.11||23.12.11||Tina Gregorič||Ana Potočnik
|-
| Jana Verbančič||A balanced mind||09.12.11||16.12.11||23.12.11||Alenka Mikuž||Ana Remžgar
|-
| Karmen Hrovat||The thread of life||14.12.11||21.12.11||04.01.12||Iza Ogris||Taja Karner
|-
| Andrej Vrankar||The things we forget||16.12.11||23.12.11||06.01.12||Jernej Mustar||Maša Mohar
|-
| Teja Banič||Cool news||16.12.11||23.12.11||06.01.12||Karmen Belšak||Jernej Mustar
|-
| Špela Pohleven||The making of crooked||16.12.11||23.12.11||06.01.12||Mitja Crček||Maša Mirković
|-
| Sabina Mavretič||A short story||21.12.11||04.01.12||11.01.12||Rok Vene||Sabina Mavretič
|-
| Karmen Belšak||Another dark horse||23.12.11||06.01.12||13.01.12||Urška Rauter||Sara Draščič
|-
| Iza Ogris||Love,love, love...||23.12.11||06.01.12||13.01.12||Ana Potočnik||Matja Zalar
|-
| Monika Škrjanc||The greenest of us all||23.12.11||06.01.12||13.01.12||Rok Štemberger||Tjaša Goričan
|-
| Ana Potočnik||Skin-deep||04.01.12||11.01.12||18.01.12||Matja Zalar||Ula Štok
|-
| Urška Rode||Smart sweat||06.01.12||13.01.12||20.01.12||Urška Navodnik||Alja Zottel
|-
| Ime in priimek||Naslov seminarja||06.01.12||13.01.12||20.01.12||||
|}

== Gradivo za seminarje ==
NOVO Gradivo za predavanja in seminarje najdete na http://bio.ijs.si/~zajec/bio2/
username: bio2
password: samozame

==Naloga==
'''Vaša naloga za seminar je: '''
Samostojno pripraviti seminar o enem od proteinov opisanih v [http://web.expasy.org/spotlight/back_issues/2011/ ProteinSpotlight] Poiskati morate vsaj še tri znanstvene članke, ki se nanašajo na opisano temo in jih uporabiti kot podlago za seminarsko nalogo!

V okviru seminarske naloge morate opraviti še naslednje naloge, katerih rešitve predložite na dodatni strani seminarske naloge, ki se ne šteje v kvoto obsega seminarja:

* sekvenca proteina in [http://www.uniprot.org/ UniProt] oznaka proteina
* slika strukture proteina (če je le-ta znana), ki jo naredite sami s programom Pymol. Če struktura še ni znana, vključite sliko proteina, ki je vašemu najbolj podoben po sekvenci in katerega struktura je znana
* poiskati morate, na katerem kromosomu se v človeškem genu nahaja ta protein in narisati shematsko sliko gena (eksonov in intronov) tega proteina. Če protein ni človeškega izvora, poiščite protein, ki je vašemu najbolj podoben in vse navedeno opišite za ta protein.

Za pripravo seminarja velja naslednje: 
* [[BIO2 Povzetki seminarjev 2011|Povzetek seminarja]] opišete na wikiju v približno 200 besedah - najkasneje do dne ko morate oddati seminar recenzentom.
* Povezavo do povzetka vnesete v tabelo seminarjev tekočega letnika.
* Seminar pripravite v obliki seminarske naloge na ~5-9 straneh A4 (pisava 12, enojni razmak, 2,5 cm robovi; važno je, da je obseg od 2700 do 3000 besed), vsebovati mora najmanj tri slike. Slika mora imeti legendo in v besedilu mora biti na ustreznem mestu sklic na sliko.
* Seminar oddajte do datuma oddaje, ki je naveden v tabeli vsakemu od recenzentov in docentu (docentu ga pošljite po e-pošti).
* Recenzenti do dneva določenega v tabeli določijo popravke in podajo oceno pisnega dela.
* Ustna predstavitev sledi na dan, ki je vpisan v tabeli. Za predstavitev je na voljo 20-30 minut. Recenzenti morajo biti na predstavitvi prisotni.
* Predstavitvi sledi razprava. Recenzenti podajo oceno predstavitve in postavijo najmanj dve vprašanji.
* Na dan predstavitve morate docentu oddati končno (popravljeno) in natisnjeno verzijo seminarja v enem izvodu.
* Seminarska naloga in povzetek morajo biti v slovenskem jeziku!

==Ocenjevanje seminarjev==
Recenzenti ocenijo seminar tako, da izpolnijo [https://docs.google.com/spreadsheet/viewform?formkey=dG1Pa3p2NXE2Vm1zX3FpVTZCT2dHVnc6MA recenzentsko poročilo] na spletu.

== Mnenje o predstavitvi ==
Vsak posameznik '''mora''' oceniti seminar, tako da odda svoje [https://docs.google.com/spreadsheet/viewform?formkey=dFNXUDBCRVBaVExvOFVxakpJUHRnOEE6MA mnenje] najkasneje v šestih dneh po predstavitvi. Kdor na seminarju ni bil prisoten, mnenja '''ne sme''' oddati.

==Urejanje spletnih strani na wikiju==
Wiki so razvili zato, da lahko spletne vsebine ureja vsakdo. Ukazi so preprosti, dokler si ne zamislite česa prav posebnega. Vseeno pa je Word v primerjavi z wikijem pravo čudežno orodje... Če imate težave z oblikovanjem besedila, si preberite poglavje o urejanju wiki-strani na Wikipediji ([http://en.wikipedia.org/wiki/Help:Editing tule] v angleščini in [http://sl.wikipedia.org/wiki/Wikipedija:Urejanje_strani tu] v slovenščini). Pomaga tudi, če pogledate, kako je zapisana kakšna stran, ki se vam zdi v redu: kliknite na zavihek 'Uredite stran' in si poglejte, kako so vpisane povezave, kako nov odstavek in podobno. ''Na koncu seveda pod oknom za urejanje kliknite na 'Prekliči'.''

==Citiranje virov==
Citiranje je možno po več shemah, važno je, da se v seminarju držite ene same.
Temeljno načelo je, da je treba vir navesti na tak način, da ga je mogoče nedvoumno poiskati.
Za citate v naravoslovju je najpogostejše citiranje po pravilniku ISO 690. [http://www.zveza-zotks.si/gzm/dokumenti/literatura.html Pravila], ki upoštevajo omenjeni standard, so pripravili pri ZTKS. Sicer pa ima vsaka revija lahko svoj način citiranja, ki ga je treba pri pisanju članka upoštevati. 
'''Citiranje knjig:''' 
Priimek, I. ''Naslov''. Kraj: Založba, letnica. 
Priimek, I. ''Naslov: podnaslov''. Izdaja. Kraj: Založba, letnica. Zbirka, številka. ISBN. 

Boyer, R. ''Temelji biokemije''. Ljubljana: Študentska založba, 2005. 
Glick BR in Pasternak JJ. ''Molecular biotechnology: principles and applications of recombinant DNA''. 3. izdaja. Washington: ASM Press, 2003. ISBN 1-55581-269-4. 
Če so avtorji trije, je beseda in med drugim in tretjim avtorjem. Če so avtorji več kot trije, napišemo samo prvega in dopišemo ''et al''. (in drugi, po latinsko). Vse, kar je latinsko, pišemo poševno (npr. tudi imena rastlin in živali, pojme ''in vivo'', ''in vitro'' ipd.).

'''Citiranje člankov:''' 
Priimek, I. Naslov. ''Naslov revije'', letnica, letnik, številka, strani. 
Lartigue C. ''et al''. Genome transplantation in bacteria: changing one species to another. ''Science'', 2007, letn. 317, str. 632-638.

Alternativni način citiranja (predvsem v družboslovju) je po pravilih APA, kjer članke citirajo takole: 
Priimek, I. (letnica, številka). Naslov. Naslov revije, strani. 
Lartigue C. ''et al.'' (2007, 317) Genome transplantation in bacteria: changing one species to another. ''Science'', 632-638.

Revija Science uporablja skrajšani zapis: 
C. Lartigue ''et al''. Science 317, 632 (2007) 

V diplomah na FKKT je treba navesti vire tako, da izpišete tudi naslov citiranega dela in strani od-do (ne samo začetne).

'''Citiranje spletnih virov:''' 
Priimek, I. ''Naslov dokumenta''. Izdaja. Kraj: Založnik, letnica. Datum zadnjega popravljanja. [Datum citiranja.] spletni naslov 
strangeguitars. ''On the brink of artificial life''. 6. 10. 2007. [citirano 13. 11. 2007] http://www.metafilter.com/65331/On-the-brink-of-artificial-life 
Navedemo čim več podatkov; pogosto vseh iz pravila ne boste našli.

BIO2 Povzetki seminarjev 2011

2011-11-18T13:31:32Z

EvaKnapič:

== Sara Draščič: On the spur of a whim ==

Serotonin ali 5-hidroksitriptamin (5-HT) spada v skupino heterogenih biokemičnih snovi, ki prenašajo informacije po živčnem sistemu in ki jim rečemo nevrotransmiterji. Ima pomembno vlogo pri veliko najrazličnejših reakcijah v telesu. Njegovo nepravilno delovanje vpliva na počutje, apetit, slabost, spanje, telesno temperaturo, staranje, bolečino, anksioznost, agresijo, spomin, migrene in na številne druge procese v organizmu. Večina serotonina se sintetizira v prebavnem traktu, preostali del pa v centralnem živčnem sistemu in trombocitih. Kljub temu, da se sintetizira le v določenih delih telesa, je prisoten povsod. Dokaz za njegovo prisotnost pa so serotoninski receptorji. Serotonin ima veliko receptorjev, ki so jih organizirali v sedem skupin glede na njihove fiziološke in strukturne razlike. Ravno zaradi tako velikega števila raznoraznih receptorjev, je serotonin pomemben pri tolikih različnih procesih, saj je njegovo delovanje, v veliki meri, odvisno od tega, na kateri receptor se bo vezal. Veliki pomen pri delovanju serotonina ima tudi njegov transporter. To je protein, katerega struktura še ni znana, vendar vemo kje in na katerem kromosomu se nahaja. Transporter je tudi glavna tarča raznih antidepresivov in drog kot so ecstasy, kokain in LSD.

== Ula Štok: Neuregulin 1 ==

Neuregulin-1 je član proteinov iz družine neuregulinov in je kodiran s strani gena NRG1. Obstaja veliko tipov Neuregulina-1, ki se razlikujejo po funkcionalnosti ter mestu v telesu na katerem delujejo. Najpogosteje delujejo v živčnem sistemu, kjer lahko z nepravilnim delovanjem med drugimi povzročajo tudi zelo razširjeno bolezen - shizofrenijo. Delujejo pa tudi na ostalih tkivih in organih (na primer: srce, pljuča, oprsje in želodec). Generalno obstajata dve poti signaliziranja Neuregulina-1, in sicer: Običajna ter neobičajna pot. Pri običajni poti je ErbB receptor aktiviran direktno, v enem koraku z vezavo Neuregulina-1. To najpogosteje povzroči dimerizacijo ali heterodimerizacijo ErbB receptorja. Dimerizacija ali heterodimerizacija sicer nista nujno potrebni, a vendar do njiju pride na skoraj vseh receptorjih ErbB. Ta združitev povzroči avto- in trans-fosforilacijo intracelularnih domen tega receptorja, kar aktivira vse nadaljnje poti signaliziranja. V končni fazi pa NRG1/ErbB signaliziranje vpliva direktno na transkripcijo. Pri neobičajni poti je postopek podoben, a vendar poteka začetna stopnja malo drugače. Na začetku namreč sodeluje JMa oblika receptorja ErbB4, ki se pod vplivom TACE cepi. Del receptorja (ErbB4-CTF) se odcepi v notranjost celice. Ta peptid je velik približno 80 kD in ima specifično izoblikovano vezavno mesto za Neuregulin-1. Nadaljnji procesi pa potekajo zelo podobno kot pri običajni signalni poti. Neuregulin-1 lahko povzroča shizofrenijo na različne načine, saj sodeluje pri zelo pomembnih procesih, kot so: tvorba sinaps, mielinizacija aksonov, razvoj oligodendrocit itd. Shizofrenija je zelo razširjena bolezen in nihče še ni odkril direktnega postopka k popolni odpravi te bolezni. A vendar, v letu 2009 se je zgodila neke vrste prelomnica v študiju shizofrenije. Odkrili so namreč, da posamezniki, ki so imeli gen za shizofrenijo niso zboleli. Še več! Napaka se jim je odrazila kot zvišanje kreativnih sposobnosti na znanstvenem ali umetniškem področju, odvisno od posameznika. Ob tem se je pojavilo mnogo vprašanj, saj bi na ta način mogoče lahko poiskali pot, da bi shizofrenija postala popolnoma ozdravljiva. A vendar, je to področje še raziskano, saj znanstveniki ne vedo po kakšnih poteh pride do tega, da te mutacije na NRG1 genu ne izrazijo v bolezenskem stanju.

== Maša Mirković: Proteinski produkti genov za disleksijo in z disleksijo povezane motnje ==

Disleksija je motnja, ki se kaže v nesposobnosti branja oziroma razumevanja prebranega, ter napakah in težavah pri izgovarjanju besed. Disleksiki,kot imenujemo posameznike, ki trpijo za disleksijo, imajo kljub normalnim intelektualnim sposobnostim, znanjem in izobrazbo, moteni veščini pisanja in branja s tendenco, da pomešajo med seboj črke ali besede med branjem ali pisanjem. V zadnjih letih, so uspeli ugotoviti mesta na kromosomih, povezana z dovzetnostjo za disleksijo. DYX1C1,KIAA0319,DCDC2 in ROBO1, so bili označeni kot kandidati, z dovzetnostjo za disleksijo. Najbolj obetaven je protein KIAA0319. Je transmembranski protein iz desetih transmembranskih vijačnic, najden v plazemski membrani nevronov. Njegov C-terminalni konec gleda v ekstracelularni matriks, manjši N-terminalni konec pa prehaja v citoplazmo nevrona. C-terminalni konec je visoko glikoziliran in nosi 5 PKD(polycystyc kidney desease) domene in eno MANEC(motif at the N terminus with eight cysteines) domeno. KIAA0319 igra vlogo pri rasti možganov in njihovi migraciji med razvojem možganov-iz tega je razvidno, da je disleksija problem v razvoju nevronov že v zgodnjih letih. Posamezniki z disleksijo nosijo izoobliko tega proteina, ki povzroči nižjo izraženost le tega. Spremembe so v 5'-regiji, ki kodira izoobliko proteina. Najopaznejše povezave z disleksijo se kažejo v 2,3 kb regiji, ki zavzema promotor, prvi nepreveden ekson in del prvega introna – odprti kromatin. Te ugotovitve vodijo, da je 5'-regija KIAA0319 gena tista lokacija alelov, ki največ prispeva k motnji branja.

== Katra Koman: INZULIN ==

Inzulin je peptidni hormon, ki sodeluje v uravnavanju ravni glukoze v krvi. Sintetizira in skladišči se v β-celicah Langerhansovih otočkov trebušne slinavke. Sinteza poteka od prekurzorske molekule preproinzulina preko proinzulina do dokončne zrele molekule inzulina, ki se shrani v skladiščnih veziklih. Ob povišanju ravni glukoze v krvi, na primer po obroku, glukoza, ki je tudi glavni stimulator sekrecije inzulina, iz krvi preide v β-celice skozi GLUT2 transporter. Tam se fosforilira v glukozo-6-fosfat, saj tako fosforilirana ne more več iz celice, lahko pa vstopi v proces glikolize, ki mu sledita še Krebsov cikel in oksidativna fosforilacija, ki povzroči pretvorbo ADP v ATP molekule. ATP molekula stimulira zaprtje kalijevih kanalčkov, kar privede do depolarizacije celične membrane, to pa sproži na odprtje kalcijevih kanalčkov in vdor Ca2+ ionov. Povišana koncentracija kalcijevih Ca2+ ionov v celici stimulira prenos in zlitje skladiščnih veziklov z inzulinom z membrano. Inzulin se tako sprosti v krvni obtok in potuje do tarčnih celic, ki imajo na površini izražene inzulinske receptorje. Ko se veže nanj, prenese signal o povišanju ravni glukoze v krvi v celico. To povzroči kaskado reakcij znotraj celice, ki pa na koncu privedejo do translokacije veziklov z GLUT4 transporterjev na površino celice. Število teh transporterjev za glukozo se na površini celične membrane poveča in glukoza lahko prehaja v celico, posledično pa pade raven glukoze v krvi. Razgradnja inzulina poteka v jetrih in ledvicah. Okvare na katerikoli stopnji poti inzulina se odražajo v diabetesu ali drugih boleznih.

== Rok Štemberger: Protein GABAA (gama aminomaslena kislina A) - zgradba, vloga in zanimivosti ==

V svoji seminarski nalogi sem raziskoval vlogo, pomen in zanimivosti proteina GABAA (gama-aminomaslena kislina A). To je receptor, ki se nahaja predvsem v centralnem živčnem sistemu in je zadolžen zato, da opravlja funkcijo inhibitorja. Lociran je na površini nevrotičnih sinaps in prekinja elektrokemični signal, tako da omogoči prehod kloridnih ionov znotraj celice. To se zgodi takrat ko se ustrezen ligand Gama veže na aktivno mesto tega receptorja. Konformacija podenot se spremeni in to omogoči aktivacijo receptorja. Znanstveniki so ugotovili, da obstaja več vrst GABAA receptorjev, kar pa je odvisno od sestave podenot. Najbolj pogoste podenote so alfa beta in gama v razmerju 2:2:1. V primeru da do prekinitve ne pride se lahko pojavijo epileptični napadi, psihiatrične motnje itd. Stres lahko v dobi odraščanja močno vpliva na GABAA receptorje in jih tudi permanentno strukturno spremeni, kar pa lahko kasneje v našem življenju vpliva predvsem na naš spanec in njegovo kvaliteto. Absint je bila v preteklosti prepovedana pijača, saj je povzročala razna obolenja zaradi substance imenovane tujon. Le ta se je vezala na GABAA receptorje in tako onemogočila njegovo delovanje, zato ker je preprečevala prehod kloridnih ionov v membrano. Sedaj potekajo raziskave teh receptorjev, saj je ključnega pomena čim boljša ozdravitev bolezni, ki nastanejo zaradi nepravilnega delovanja GABAA receptorja.

== Veronika Jarc: Perforin ==

Perforin je protein, ki nastane iz citotoksičnih limfocitov T. S pomočjo grancimov napade tarčno celico in jo uniči. Rečemo lahko, da je pomemben člen pri imunskem odzivu in sodeluje s NK celicami. Sestavljen je iz 555 aminokislin, njegova molekulska masa pa je 62-67 kD. Sestavljen je iz dveh pomembnih domen, domene MACPF in domene C2. Za domeno C2 je značilno, da ima afiniteto do Ca2+ ionov. Saj se na lipidni dvosloj veže le ob prisotnosti kalcija. Drugače obstajata dva različna tipa C2 domene, ki sta bila izolirana iz različnih organizmov. Lahko rečemo, da sta oba tipa zelo podobna v tem, da sta pri tipu 1 N-konec in C-konec obrnjena na vrh domene, kar je nasprotno kot pri tipu 2. Poznamo tri MACPF domene: Plu-MACPF, C8a MACPF in lipokalin C8g. Vse te domene primerjamo z skupino proteinov citolizinov in ugotovimo nekaj podobnosti in nekaj razlik. Na splošno, pa lahko rečemo, da je evolucija poskrbela tako, da so sta si domena MACPF in citolizini raszlični le v nekaj aminokislinah. Poznamo tri mehanizme kako perforin preide v tarčno celico in pri tem pomaga gramcimom B uničit to celico. Prvi mehanizem je prehajanje preko perforinske pore in sicer s pomočjo veziklov preide v celico. Naslednji mehanizem je endosomolitični model, pri katerem je pomemben kompleks s pomočjo katerega prehaja v celico. Kot zadnji mehanizem pa je model prehodne perforinske pore, ki pove, da perforin tvori kanalčke s pomočjo katerih grancimi B preidejo direktno v celico. Grancimi B so serinske proteaze, ki se sintetizirajo v citotoksičnih limfocitih T in NK celicah.

== Taja Karner: Glavoboli in migrene ==

Zaradi stresnega in hitrega tempa življenja, vse več ljudi trpi za občasnimi glavoboli, ki so najpogosteje posledica utrujenosti. Prav tako je vedno več ljudi, ki trpijo za močnejšimi oblikami glavobolov imenovanih migrene. V hujših oblikah migrene lahko glavobol traja do dva dni, močno migreno lahko spremljajo še drugi simptomi kot so slabost, bruhanje, občutljivost na svetlobo in močan zvok, depresija ter nespečnost. Mutacija, ki je največji krivec za nastanek bolezni se pojavlja na kromosomu 10 na genu KCNK18. Ta zapisuje protein TRESK, ki se nahaja v hrbtenjači in deluje kot kalijev kanalček. Mutacija povzroči, da ne pride do izmenjavanja ionov, kar povzroči hude glavobole. V raziskavah so odkrili zanimivo povezavo z anestetikom. Ta namreč ne glede na mutacijo ponovno aktivira kanal. To bi lahko učinkovito pozdravilo migrene, če bi ga le uspeli spraviti v primerno obliko. Ugotovili so tudi, da zdravila, ki vsebujejo citosporin in takrolimus v večini primerov povzročajo migrene v zdravstvu pa jih še vseeno pogosto uporabljajo. Odkritje te mutacije predstavlja revolucijo v zdravstvu in verjamem, da bo kmalu vodilo do odkritja učinkovitega zdravila proti migrenam.

== Ana Dolinar: Univerzalna kri – prihodnost transfuzijske medicine? ==

α-galaktozidaza (AGAL_HUMAN) je glikozil-hidrolazni encim. Spada v GH27-D (klan D, 27. družina) in ima aktivno mesto v obliki (β/α)8 sodčka. Encim zapisuje gen GLA, ki se nahaja na kromosomu X.

Ideja o univerzalni krvi, ki bi bila primerna za transfuzijo, ne glede na krvno skupino pacienta, je med znanstveniki prisotna že približno trideset let.
Razvili so tri metode za pretvorbo različnih antigenov v antigen 0 (po sistemu AB0), ki je primeren za transfuzijo v vse krvne skupine.
:#Encimska razgradnja antigenov A in B do antigena 0. Za antigene A so uporabili α-N-acetilgalaktozaminidazo, vendar so antigeni preveč kompleksni in metoda ni bila uspešna. Pri antigenih B so dosegli popolno pretvorbo v antigen 0 z uporabo α-galaktozidaze iz bakterije ''Streptomyces griseoplanus''.
:#Prekrivanje površine eritrocitov z maleimidofenil-polietilen-glikolom (Mal-Phe-PEG). Prekrije vse antigene, ne samo A ali B, vendar metoda ni uspešna, ker polietilen-glikol povzroča imunski odziv.
:#Pridobivanje univerzalnih rdečih krvnih celic iz pluripotentnih matičnih celic. Uspeli so pridobiti zrele eritrocite, ki so popolnoma funkcionalni.
Uporaba univerzalne krvi bi zmanjšala ali celo izničila imunski odziv ob transfuziji, prav tako ne bi bilo možnosti za transfuzijo napačne krvne skupne zaradi človeške napake. Metode trenutno niso dovolj izpopolnjene, da bi bilo možno pričakovati njeno uporabo v bližnji prihodnosti.

== Maša Mohar: Moški ali ženska to je sedaj vprašanje?(SRY - faktor za določitev spola) ==

SRY gen kodira Sry protein ki je član družine Sox (Sry related HMG box) transkripcijskih faktorjev. Poznamo jih okoli 20 pri človeku in miškah ter še mnogo drugih. Sox proteini imajo zelo različne vloge v embriogenezi in pri razvoju mnogih drugih organov. Tipično delujejo tako kot nekakšna stikala v diferenciaciji celic- sprožijo razvoj določenih celic. Sry je prav tako kot ostali člani te družine karakteriziran po HMG( high mobility group). HMG je drugače skupina specifičnih transkripcijskih faktorjev, ki imajo ~ 80 AK dolge strukturalno podobne domene za vezavo na DNA. Te domene oz. domena če je samo ena se veže na zaporedje (A/T)ACAA(T/A) v majhni žleb DNA. S tem ustvari zvitje DNA za približno 60- 85 stopinj. S tem ko se DNA zvije se razkrijejo mesta za izražanje drugih genov, recimo Sox9, ki kodira Sox9 protein ki pomaga pri diferenciaciji Sertoli celic in tako pri oblikovanju testisov, s tem pa determinira moški spol. Ugotovili smo tudi da obstaja veliko genskih bolezni povezanih s Sry genom in da lahko obstaja tudi ženska z XY spolnima kromosomoma, ker se pri njej zaradi mutacij Sry protein ne izrazi, prav tako pa obstajajo tudi moški z XX spolnima koromosomoma, kjer se enem od X kromosomov lahko izrazi SRY gen ob nepravilnostih pri očetovem delu zapisa. V bistvu sem prišla do zaključka da je zelo tanka meja med moškim in ženskim oblikovanjem spola, ena majhna mutacija oz. ena majhna razlika lahko privede do nastanka ženske ali moškega.

== Urška Rauter: A Green Glow: zgradba in funkcija encima luciferaze ==
Luciferaza je encim odvisen od ATP in magnezijevih ionov. Proces bioluminiscence se začne z vezavo na substrat luciferin, tvori se adenilatni intermediat in ob prisotnosti molekularnega kisika izhaja svetloba. Luciferaza je zgrajena iz dveh ločenih domen, večja se nahaja na N-koncu in manjša na C-koncu molekule, večja domena pa ima tudi svoje poddomene. Domeni sta med seboj ločeni z razpoko, kjer naj bi se po domnevanjih nahajalo tudi aktivno mesto encima. Luciferaza predstavlja tudi nov način mehanizma tvorbe adenilatnega intermediata med encimi in ponuja razlago za marsikatero metabolično pot.
Velika dilema, ki me med znanstveniki ostaja pa je razlika v barvi svetlobe, ki jo proces oksidacije luciferina emitira. Najverjetneje je za to odločilna keto tavtomerna oblika oksiluciferina in tudi resnonančna stabilizacija njegovega fenolatnega aniona, čeprav so znanstveniki odkrili tudi veliko drugih možnih vzrokov za različne barve (različne aminokisline, polarnost okolja, pH, ...).
Luciferaza se veliko uporablja v medicini, kjer služi kot marker molekul v telesu in tako pripomore k boljšem razumevanju različnih bolezni in infekcij, kot tudi sami strukturi celic in njenih organelov.

== Mirjam Kmetič: Mint condition (limonen-3-hidroksilaza in limonen-6-hidroksilaza) ==

Klasasta meta vsebuje encim limonen-6-hidroksilazo, ki sodeluje pri pridobivanju karvona. Poprova meta pa vsebuje limonen-3-hidroksilazo, ki je udeležena pri proizvodnji mentola. Obe hidroksilazi pripadata družini citokromov P450, njeni predstavniki pomembno sodelujejo pri proizvajanju različnih oksidiranih monoterpenov, ki so vir arom eteričnih olj. Karvon in mentol sta končna produkta hidroksilacije limonena. Ta encima sta si zelo podobna in njuni vezavni mesti za substrat sta zelo omejeni. Velja pravilo, da za spremembo aktivnosti v družini citokromov P450 potrebujemo določeno število mutacij, vendar je za modifikacijo vezavne aktivnosti limonenovih hidroksilaz potrebna samo ena. Ta fenilalanin v izolevcin mutacija povzroči, da se limonen-6-hidroksilaza spremeni v limonen-3-hidroksilazo! Mutiran encim je tako sposoben sinteze mentola tako kot encim v poprovi meti! Taka mutacija kaže, da sta prav ti dve aminokislini ne le nujni, temveč tudi prav zagotovo vpleteni pri orientaciji limonena v aktivnem mestu tako, da se ta hidroksilizira na ali C3 ali C6 poziciji. Posamične mutacije, ki lahko drastično spremenijo funkcijo proteina, so znanstveno zanimive. Nakazujejo ne le na zelo specifične manjše regije v sekvenici proteina, temveč so tudi ključne za razumevanje področij, kot so vezava in orientacija substrata, funkcija encima, metabolična pot in struktura vezavnega mesta.

== Sandi Botonjić: Kokain esteraza ==

Znanstveniki so v rizosferi kokinih plantaž (Erythroxylum coca) našli sev MB1, gram pozitivne bakterije Rhodococcus sp.. Tej bakteriji kokain predstavlja glavni vir ogljika in dušika in zato so znanstveniki izolirali osrednji encim njenega metabolizma tj. kokain esterazo (v nadaljevanju cocE). Encim je sestavljen iz treh domen: DOM1, ki vsebuje nabor kanoničnih α-vijačnic in β-ploskev; DOM2 - domena le z α-vijačnicami; in DOM3 je roladi podobna struktura z β-ploskvami. CocE je serinska esteraza, katere aktivno mesto se nahaja na stičišču vseh treh domen. Ta hidrolizira kokain na ekgonil metil ester in benzojsko kislino, ki nimata psihoaktivnih učinkov. CocE je pravi Ferrari v primerjavi z drugimi esterazami, saj lahko razgradi enako količino kokaina 1000 krat hitreje. Tako lahko postane neprecenljiva pri nujnih intervencijah v primeru prevelikega odmerka, saj bi intravenozni vbrizg cocE močno zmanjšal razpolovni čas kokaina. CocE je predmet številnih raziskav, v katerih znanstveniki proučujejo njeno termostabilnost in njenih mutiranih oblik, saj njen razpolovni čas pri fiziološki temperaturi traja le nekaj minut. Znanstveniki pa na podlagi ugotovitev iz raziskav cocE razvijajo tudi učinkovita protitelesa z vsaj podobnimi katalitičnimi parametri, ki bi brez imunskega odziva odlično delovala v bioloških sistemih.

==Tjaša Flis: Parkinsonizem in Parkin protein==

Parkinsonova bolezen je vse pogostejša bolezen pri starostnikih, njeni simptomi pa so tresavica, mišična otrdelost in upočasnjena motorika. Vzrok se skriva v propadu dopamnergičnih nevronskih celic. Bolezen je lahko avtosomno dominantno dedovana, kar pomeni, da pacienti podedujejo eno normalno in eno mutirano kopijo gena. Slednja prevladuje in se deduje naprej. Pri Parkinsonovi bolezni se mutacija zgodi v Park2 genu, ki kodira Parkin protein ali E3 ubikvitin ligazo. Parkin na poškodovane ali na preveč izražene proteine pripne ubikvitin (označevalni protein), ki jih nato usmeri v proteasom, to je velik razgradni kompleks v celicah.
Če mutacija poškoduje Parkin, je pot razgradnje onemogočena, to pa pomeni, da se v celici akumulirajo odvečni proteini. Tvorijo se Lewy-eva telesca polna teh proteinov, ki nadomestijo celične organele v nevronskih celicah, kar vodi do prenehanja njihovega delovanja. Ker pa ima Parkin več kot samo en substrat ki ga ubikvitinira, je točen mehanizem bolezni še dandanes uganka.
Eden izmed najbolj poznanih substratov je transmembranski protein Pael-R. Zvitje tega proteina poteka ob prisotnosti šaperonov. Prevelika koncentracija tega receptorja lahko izzove stres v endoplazmatskem retikulumu situiranem v nevronskih celicah. V primeru da je Parkin neaktiven, Pael-R povzroči celično smrt. Vendar to je le ena izmed možnih rešitev, substratov je namreč vsaj še dvajset, raziskave pa se nadaljujejo.

== Matja Zalar: Vloga SRK in SCR proteinov pri preprečevanju incestnega razmnoževanja cvetočih rastlin ==

Rastline so za zaščito pred samooplojevanjem razvile več vrst mehanizmov prepoznavanja lastnega peloda na molekularni ravni. Pri cvetočih rastlinah je najpogostejši mehanizem tipa SSI ali sporofitične lastne inkompatibilnosti. Pri družini ''Brassicaceae'' je za aktivacijo SSI ključna interakcija med transmembranskim proteinom SRK, ki predstavlja žensko determinanto odziva, in njenim ligandom - proteinom SCR, drugače imenovanim tudi moška determinanta odziva na lastno inkompatibilnost. Specifičnost vezave je zagotovljena s polimorfizmom alel obeh determinant. V posameznih vrstah je možno najti tudi do 100 različnih S-haplotipov genov za determinanti.
Vezava liganda na receptor bo uspešna le, če oba izhajata iz istega S-haplotipa. Vezava SCR na zunajcelično, N-glikolizirano domeno SRK povzroči nastanek kompleksa treh proteinov, ki s svojo aktivnostjo sproži kaksado reakcij, kar v končni fazi pripelje do preprečitve samooploditve.
Na neugodne življenske pogoje, ki so onemogočali medsebojno opraševanje, so se nekatere rastline prilagodile s favorizacijo samooplojevanja. Pri njih so mutacije S-lokusa, ki nosi zapis za SRK in SCR, povzročile nepravilno delovanje SI ali njegovo popolno odpoved. To pa seveda vodi v neprepoznavanje lastnega peloda in rastlina se samooprašuje. Najbolj znan primer take rastline je ''Arabidopsis thaliana'', ki se zaradi svojih specifičnih lastnosti uporablja kot modelni organizem v številnih študijah lastne inkompatibilnosti.

== Matevž Ambrožič: BSX protein in debelost ==

Za primeren občutek sitosti ali lakote glede na stanje energetskih zalog v telesu in odgovarjajoč vnos hrane ter porabo energije je odgovorna zapletena pot sporočanja. Začne se s tremi hormoni: inzulin, leptin in grelin. Leptin in inzulin se sprostita, ko so maščobne in hidratne zaloge v telesu polne in morata do možganov prenesti signal za prenehanje hranjenja, grelin pa ravno nasprotno. Vsi po krvi potujejo do hipotalamusa, predela možganov, ki je odgovoren za energijsko ravnovesje. V hipotalamusu sta dva tipa živčnih celic: oreksigene in anoreksigene. Prve sproščajo NPY in AgRP, nevropeptida, ki spodbujata hranjenje in zmanjšata porabo energije, druge pa α-MSH in CART, katerih učinek je nasproten. Našteti nevropeptidi se iz nevronov sprostijo po vezavi ustreznega izmed treh hormonov in prenesejo signal naprej, do končne spremembe v vnosu ali porabi energije. Glavni protein seminarja, BSX (brain specific homeobox) protein je transkripcijski faktor, ki spodbudi ekspresijo genov za AgRP in NPY, hkrati pa je odgovoren za premik organizma v iskanju hrane. Če v opisanem sistemu pride do napake, so pojavi nepotreben občutek lakote, kar je vzrok mnogih primerov debelosti. V boju z bolezensko debelostjo so ključne raziskave na BSX proteinu, saj je osrednji člen poti, ki v možgane prenese (včasih lažen) občutek lakote.

== Kaja Javoršek: A grey matter ==

Mikrocefalin je protein, ki ga kodira enakoimenski gen. Mikrocefalin naj bi kontroliral poliferacijo in diferenciacijo nevroblastov med nevrogenezo. Odkritje, da je mikrocefalin odločilen regulator velikosti možganov, je sprožilo hipotezo, da je igral vlogo v evoluciji možganov.
Razen v možganih najdemo mikrocefalin tudi v ledvicah, srcu, pljučih, vranici in skeletnih mišicah. Vendar pomen mikrocefalina v teh organih še ni znan.
Mutacije na genu mikrocefalina vodijo do nastanka mikrocefalije. To je bolezen razvoja živčnega sistema in je definirana kot resno zmanjšana velikost možganov. Pri odraslih je normalen volumen možganov od 1200 cm3 do 1600 cm3, pri odraslih s primarno mikocefalijo pa okoli 400 cm3 . Poleg mirocefalina pa povzročajo mikrocefalijo še mutacije petih genih (ASPM, MCPH2, CDK5RAP2, MCPH4, CENPJ)
Mikrocefalin ima tri BRCT domene na C – koncu. BRCT domene so prisotne v veliko ključnih proteinih, ki kontrolirajo delitev celice. Zato predvidevajo da mikrocefalija nastane, ker je ovirana normalna regulacija delitve celic v možganih.
Ugotovili so, da je protein mikrocefalin dol 835 aminokislin. Zaradi mutacije na genu mikrocefalina se ta protein skrajša na 25 aminokislin.
Znanstveniki so izvedli raziskavo ali gena mikrocefalin in ASPM vplivata na inteligenco. Na podlagi treh raziskav so zaključili, da inteligenca ni povezana z dominantnimi aleli ASPM – ja ali mikrocefalina.

== Rok Vene: A mind astray ==

Alzheimerjeva bolezen postaja vedno bolj aktualna tematika. Trenutno je na svetu več kot 26 milijonov ljudi s to obliko demence. Zaradi daljše življenjske dobe pa pričakujemo, da bo število obolelih samo še naraščalo. Alzheimerjeva bolezen prizadene centralni živčni sistem, v možganih se nalagajo snovi, ki povzročijo propad živčnih celic. Ena izmed snovi, ki se nalagajo v možganih so nefunkcionalni Tau proteini. Tau proteini sodijo v družino proteinov imenovanih microtubule-associated proteins (MAP), njihova naloga pa je je stabilizacija mikrotubulov. To dosežejo tako, da se na mikrotubule vežejo. Poleg tega predvidevajo, da imajo Tau proteini še eno nalogo. Sodelovali naj bi v kompleksu za uravnavanje vzdražnosti živčnih celic. Nefunkcionalnost Tau proteinov povezujejo z različnimi boleznimi, ki jih poznamo pod skupnim imenom tauopatije. V primeru Alzheimerjeve bolezni je Tau protein nefunkcionalen, zato ker je hiperfosforiliran, kar mu onemogoča vezavo na mikrotubule. Tau proteini zato tvorijo netopne agregate – nevrofibrilarne pentlje, ki najbrž povzročijo odmiranje živčnih celic. Pri iskanju učinkovin proti hiperfosforilaciji in agregaciji Tau proteina, so znanstveniki raziskali protein FKBP52. Ta protein ima več funkcij. Osredotočili so se predvsem na njegove šaperonske lastnosti. Ugotovili so, da se FKBP52 veže na hiperfosforiliran Tau protein, in tako prepreči agregacijo Tau proteina, ki je odgovorna za odmiranje nevronov.

== Ines Šterbal: LTP1 ==

Protein LTP1, izoliran iz ječmenovega zrna, spada v družino lipidnih prenašalnih proteinov (lipid transfer protein –LTP). Je dobro topen protein, ki se nahaja v alevronski plasti ječmenovega semena. Sestavljen je iz štirih heliksov, ki so povezani z disulfidnimi mostički. Ima dobro definiran C-terminalni konec. V razmerah in vivo je globularni protein, s stožčastim hidrofobnim jedrom, ki se razteza od enega konca molekule do drugega. Sposoben je vezati različne lipide, kot so maščobne kisline ali acetil-koencim A. LTP1 proteini so na površini aktivni proteini, so stabilni, denaturirajo šele okrog 100 °C. Vloga LTP1 proteina in vivo še ni znana. In vitro je glavni protein pri penjenju piva. Opravlja pa še številne druge funkcije, odvisno od tega, kateri ligand ima vezan. LTP1 proteini so verjetno vključeni v prenos lipidov preko membrane in celo v nastanek membrane, lahko bi imeli vlogo v transportu monomera Cutin, vlogo naj bi igrali tudi v obrambnem mehanizmu rastlin. Lipidi, ki so vezani na LTP1 bi naj imeli antibakterijsko aktivnost za bakterije in glive.
Vsi podatki kažejo, da so povezave med sladkorji in proteini, ki nastanejo kot produkt Milardove reakcije, prvi korak do nastanka pivovske pene. Kaže, da je kontrola glikacije LTP1 proteinov med slajenjem in varjenjem piva, nujna za optimalno penjenje piva.

== Mitja Crček: DSIP in spanje ==

Pred 2000 leti so ljudje verjeli, da postanemo zaspani zaradi nekakšnih želodčnih hlapov, ki gredo v možgane, se tam kondenzirajo, zamašijo pore in posledično povzročajo zaspanost. Kasneje so seveda ugotovili da temu ni tako, leta 1977 pa so odkrili majhne peptide, ki naj bi nas uspavali in jih poimenovali Delta Sleep-Inducing peptide (DSIP). DSIP je majhen peptid, sestavljen iz devetih aminokislinskih ostankov in maso 850 daltonov, prvič pa so ga odkrili pri zajcih. Sodeloval naj bi tako pri endokrini regulaciji kot pri fizioloških procesih (poveča učinkovitost oksidativne fosforilacije), pomembno vlogo pa naj bi imel tudi v medicini in pri zdravljenju bolezni. Ker naj bi podaljševal REM fazo, bi ga lahko uporabljali tudi kot dodatek pri zdravljenju alkoholizma ali ga dodajali antidepresivom in pomirjevalom, ki skrajšujejo REM fazo. Raziskave so spremljale tudi vpliv DSIP-ja na nespečnost. Ugotovili so, da DSIP rahlo povečuje kvaliteto spanja in skrajšuje latenco uspavanja, na trajanje budnosti in druge parametre pa ne vpliva, zato so si strokovnjaki enotni, da ima DSIP le rahle terapevtske učinke na nespečnost. Delovanje peptida pa še vedno ni povsem razjasnjeno in le želimo si lahko, da bodo novejše raziskave prinesle nove informacije, saj ima DSIP vsekakor velik potencial v medicini.

== Dominik Kert: FOXP2, govoreči protein ==

Ljudje in živali se razlikujejo. Za znanstvenike 19. stoletja je bilo zelo fascinantno to, da mi lahko govorimo, se sporazumevamo in pomnimo besede, medtem ko živali ne morejo. Ko se je pojavila družina KE na koncu 90. let prejšnjega stoletja, so znanstveniki ugotovili, da obstaja gen, ki kodira FOXP2. Družina KE je slovi po tem, da ima polovica njenih članov težave z govorom. Tako so ugotovili, da se mutacija prenaša avtosomno in dominantno. In verjetno na to vpliva mutacija FOXP2, FOXP2 protein pa je po vsej verjetnosti odločilen faktor pri govoru.
FOXP2 protein je sestavljen iz 715 aminokislin in spada med družino transkripcijskih faktorjev, ki se imenuje FOX (zaradi 'forkhead box' domene). Zanimivo je, da se ta gen razlikuje od gena opic (šimpanz, gorila, makaki) le za dve in od miši le za tri aminokisline. To se znanstvenikom zdi zelo zanimivo, ker je verjetno zaradi teh dve sprememb v aminokislinskem zaporedju prišlo do sprememb pri sporazumevanju. Zaradi teh dejstev so se naprej usmerili na to, ali je bil gen res pod vplivom naravne selekcije in ugotovili so, da je bil res.
FOXP2 na te spremembe vpliva v možganih, je pa prisoten tudi v pljučih, drobovju in srcu. Vendar njegova funkcija tam še ni znana.

== Petra Malavašič: Ureaza bakterije Helicobacter pylori ==

Bakterija Helicobacter pylori spada med patogene mikrobe. Znanstvenika Warren in Marshall sta leta 1987 odkrila to bakterijo ter ugotovila, da je s to bakterijo povezana razjeda na želodcu. Leta 2005 sta prejela Nobelovo nagrado. Že vsak drugi človek je okužen s to bakterijo. Naseljena je na želodčni sluznici in povzroča kronično vnetje želodčne sluznice. Bakterija se lahko naseli in se razmnožuje v prisotnosti želodčne kisline, kjer je pH okoli 2. Posebni obrambni mehanizmi omogočajo bakteriji, da lahko preživi v kislem okolju. Encim ureaza je pri tem najpomembnejši. Ureaza je encim, ki katalizira hidrolizo uree, pri čemer nastane amoniak, ki se v končni fazi veže z molekulami vode v amonijev hidroksid, ki poveča pH v neposredni okolici bakterije. Encim ureaza se nahaja v citoplazmi bakterijske celice in na njeni površini. Sam encim je zgrajen zelo kompleksno in omogoča bakteriji preživetje. Posebna kompleksna zgradba encima onemogoči, da bi kislina želodčnega soka denaturirala encim. Encim sestavljata dva kompleksa (αβ) štirih prostorsko razporejenih (αβ)3 enot.

== Matevž Merljak: CEM15, VIF in infektivnost retrovirusov ==

Ena izmed komponent obrambnega mehanizma pred retrovirusi v nekaterih človeških celicah je citidinska deaminaza CEM15 (APOBEC3G). V celicah, ki jo izražajo, se retrovirusi brez posebnega proteina (VIF, “viral infectivity factor”) ne morejo uspešno množiti, zato takim celicam pravimo “nepermisivne” celice.

CEM15 deluje tako, da med procesom reverzne transkripcije v novonastali “minus” DNA verigi številne citidinske baze pretvori v uridinske, ter s tem povzroči tako zmanjšano obstojnost z uracilom bogate DNA verige, kot tudi zamenjave gvanozinskih baz z adenozinskimi v kodirajoči (“plus”) verigi DNA. Čeprav takšna hipermutacija za nadaljno infektivnost virusa ni vedno usodna (torej lahko tako mutirana DNA v nekaterih primerih še vedno tvori funkcionalne viruse), je običajno dovolj obsežna, da onemogoči uspešno reprodukcijo virusa.

Raziskave kažejo, da CEM15 ne napade nastajajoče DNA kot lasten celični odgovor na infekcijo, pač pa se med izgradnjo novih virusov vgradi v le-te ter po infekciji nove celice povzroči omenjene spremembe v nastajajoči DNA verigi.

Že omenjen faktor VIF izhaja iz virusa HIV-1, ki primarno napada sicer nepermisivne limfocite T. Naloga VIF je preprečitev vgradnje CEM15 v nastajajoče viruse, to pa doseže tako z oteževanjem njene translacije, kot tudi z indukcijo razgradnje CEM15 v proteasomu.

== Eva Knapič: TSH3 - Kaj novorojenčkom omogoča zadihati? ==

Kaj novorojenčkom omogoča zadihati? Raziskave so pokazale, da ima eno izmed vodilnih vlog pri začetku dihanja protein teashirt homolog 3 (TSH3). To je protein, ki ga uvrščamo med transkripcijske faktorje. Po strukturi spada v družino cinkovih prstov, kjer so sekundarne strukture koordinirane s cinkovim ionom. TSH3 ima pet tako urejenih struktur in vse spadajo v Cys2His2 skupino – cinkov ion koordinira dva cisteinska in dva histidinska ostanka ßßα podenote.
Organizem brez zapisa za teashirt 3 protein se v času embrionalnega razvoja navidezno ne razlikuje od organizmov, ki ta zapis imajo. Vendar so podrobnejše raziskave pokazale, da se brez prisotnosti proteina teashirt 3 dokončno ne oblikujejo pljučni mešički, ki so funkcionalna enota pljuč, saj tam poteka izmenjava plinov. Odsotnost proteina povzroča povečano apoptozo nevronov motoričnega jedra v možganskem deblu, s tem so proteinu pripisali zmožnost inhibicije apoptoze nevronov. Prav tako so nezmožnost odziva organizma na pH spremembe okolja pripisali pomanjkanju proteina TSH3.
Iz vseh teh pomanjkljivostih, ki jih povzroča TSH3 so raziskovalci prišli do zaključka, da novorojenček brez zapisa za protein ni zmožen zadihati, ker ni sposoben odziva na spremembo okolja, predvsem pH in tako ne more vzdrževati homeostaze, ki je potreba na preživetje.

BIO2 Seminar 2011

2011-10-09T11:13:35Z

EvaKnapič: /* Seznam seminarjev - datumi še niso dokončni, listka na katerem imam napisano kdaj kdo ne more nimam doma in bom to popravil v ponedeljek */

= Biokemijski seminar =

Seminarje vodi doc. dr. Gregor Gunčar in so na urniku vsako sredo in petek po eni uri predavanj iz Biokemije.

Ocena seminarjev predstavlja 30% končne ocene in vsebuje vse točke, ki jih študent/ka lahko zbere pri seminarju in ostalih dejavnostih, ki niso del pisnega izpita.

== Seznam seminarjev - datumi še niso dokončni, listka na katerem imam napisano kdaj kdo ne more nimam doma in bom to popravil v ponedeljek==
Vpišite svoj izbrani naslov!!!
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Ime in priimek'''
| align="center" style="background:#f0f0f0;"|'''Naslov seminarja'''
| align="center" style="background:#f0f0f0;"|'''Rok za oddajo'''
| align="center" style="background:#f0f0f0;"|'''Rok za recenzijo'''
| align="center" style="background:#f0f0f0;"|'''Datum predstavitve'''
| align="center" style="background:#f0f0f0;"|'''Recenzent1'''
| align="center" style="background:#f0f0f0;"|'''Recenzent2'''
|-
| Ula Štok||Tipping the mind||17.10.11||19.10.11||21.10.11||||
|-
| Maša Mirković||Naslov seminarja||17.10.11||19.10.11||21.10.11||||
|-
| Sara Draščič||Naslov seminarja||17.10.11||19.10.11||21.10.11||||
|-
| Katra Koman||Naslov seminarja||18.10.11||23.10.11||26.10.11||||
|-
| Iza Ogris||Naslov seminarja||21.10.11||25.10.11||28.10.11||||
|-
| Ana Remžgar||Naslov seminarja||21.10.11||25.10.11||28.10.11||||
|-
| Urška Rauter||Naslov seminarja||21.10.11||25.10.11||28.10.11||||
|-
| Taja Karner||Naslov seminarja||21.10.11||26.10.11||02.11.11||||
|-
| Rok Štemberger||Naslov seminarja||21.10.11||28.10.11||04.11.11||||
|-
| Maša Mohar||Naslov seminarja||21.10.11||28.10.11||04.11.11||||
|-
| Veronika Jarc||Naslov seminarja||21.10.11||28.10.11||04.11.11||||
|-
| Mirjam Kmetič||Naslov seminarja||26.10.11||02.11.11||09.11.11||||
|-
| Janez Meden||Naslov seminarja||28.10.11||04.11.11||11.11.11||||
|-
| Tjaša Flis||Naslov seminarja||28.10.11||04.11.11||11.11.11||||
|-
| Sandi Botonjić||Naslov seminarja||28.10.11||04.11.11||11.11.11||||
|-
| Kaja Javoršek||Naslov seminarja||02.11.11||09.11.11||16.11.11||||
|-
| Rok Vene||Naslov seminarja||04.11.11||11.11.11||18.11.11||||
|-
| Ines Šterbal||Naslov seminarja||04.11.11||11.11.11||18.11.11||||
|-
| Andreja Bratovš||Naslov seminarja||04.11.11||11.11.11||18.11.11||||
|-
| Matevž Ambrožič||Naslov seminarja||09.11.11||16.11.11||23.11.11||||
|-
| Matevž Merljak||Naslov seminarja||11.11.11||18.11.11||25.11.11||||
|-
| Mitja Crček||Naslov seminarja||11.11.11||18.11.11||25.11.11||||
|-
| Dominik Kert||Naslov seminarja||11.11.11||18.11.11||25.11.11||||
|-
| Petra Malavašič||Naslov seminarja||16.11.11||23.11.11||30.11.11||||
|-
| Eva Knapič||Life's first breath||18.11.11||25.11.11||02.12.11||||
|-
| Marko Radojković||Naslov seminarja||18.11.11||25.11.11||02.12.11||||
|-
| Tjaša Goričan||Naslov seminarja||18.11.11||25.11.11||02.12.11||||
|-
| Tina Gregorič||Naslov seminarja||23.11.11||30.11.11||07.12.11||||
|-
| Tamara Marić||Naslov seminarja||25.11.11||02.12.11||09.12.11||||
|-
| Ana Dolinar||The juice of life||25.11.11||02.12.11||09.12.11||||
|-
| Maja Remškar||Naslov seminarja||25.11.11||02.12.11||09.12.11||||
|-
| Matja Zalar||Naslov seminarja||30.11.11||07.12.11||14.12.11||||
|-
| Urška Navodnik||Naslov seminarja||02.12.11||09.12.11||16.12.11||||
|-
| Jernej Mustar||Silent pain||02.12.11||09.12.11||16.12.11||||
|-
| Ines Kerin||Naslov seminarja||02.12.11||09.12.11||16.12.11||||
|-
| Alja Zottel||Naslov seminarja||07.12.11||14.12.11||21.12.11||||
|-
| Alenka Mikuž||Naslov seminarja||09.12.11||16.12.11||23.12.11||||
|-
| Maja Grdadolnik||Ear of Stone||09.12.11||16.12.11||23.12.11||||
|-
| Jana Verbančič||Naslov seminarja||09.12.11||16.12.11||23.12.11||||
|-
| Petra Gorečan||Naslov seminarja||14.12.11||21.12.11||04.01.12||||
|-
| Karmen Hrovat||Naslov seminarja||16.12.11||23.12.11||06.01.12||||
|-
| Andrej Vrankar||The things we forget||16.12.11||23.12.11||06.01.12||||
|-
| Teja Banič||Naslov seminarja||16.12.11||23.12.11||06.01.12||||
|-
| Špela Pohleven||Naslov seminarja||21.12.11||04.01.12||11.01.12||||
|-
| Ime in priimek ||Naslov seminarja||23.12.11||06.01.12||13.01.12||||
|-
| Ime in priimek ||Naslov seminarja||23.12.11||06.01.12||13.01.12||||
|-
| Ime in priimek ||Naslov seminarja||23.12.11||06.01.12||13.01.12||||
|-
| Ime in priimek ||Naslov seminarja||04.01.12||11.01.12||18.01.12||||
|-
| Ime in priimek ||Naslov seminarja||06.01.12||13.01.12||20.01.12||||
|-
| Ime in priimek ||Naslov seminarja||06.01.12||13.01.12||20.01.12||||
|-
| Ime in priimek ||Naslov seminarja||06.01.12||13.01.12||20.01.12||||
|}

== Gradivo za seminarje ==
Gradivo za predavanja in seminarje najdete na http://bio.ijs.si/~zajec/bio2/
username: bio2
password: samozame

==Naloga==
'''Vaša naloga za seminar je: '''
Samostojno pripraviti seminar o enem od proteinov opisanih v [http://web.expasy.org/spotlight/back_issues/2011/ ProteinSpotlight] Poiskati morate vsaj še tri znanstvene članke, ki se nanašajo na opisano temo in jih uporabiti kot podlago za seminarsko nalogo!
V seminarsko nalogo mora biti vključeno:
* sekvenca proteina in SwissProt oznaka proteina
* slika strukture proteina (če je le-ta znana), ki jo naredite sami s programom Pymol. Če struktura še ni znana, vključite sliko proteina, ki je vašemu najbolj podoben po sekvenci in katerega struktura je znana
* poiskati morate, na katerem kromosomu se v človeškem genu nahaja ta protein in narisati shematsko sliko gena (eksonov in intronov) tega proteina. Če protein ni človeškega izvora, poiščite protein, ki je vašemu najbolj podoben in vse navedeno opišite za ta protein.

Za pripravo seminarja velja naslednje: 
* [[BIO2 Povzetki seminarjev 2011|Povzetek seminarja]] opišete na wikiju v približno 200 besedah, besedilo naj vsebuje sliko strukture proteina, ki jo sami narišete s programom PyMol - najkasneje do dne ko morate oddati seminar recenzentom.
* Povezavo do povzetka vnesete v tabelo seminarjev tekočega letnika.
* Seminar pripravite v obliki seminarske naloge na ~5-9 straneh A4 (pisava 12, enojni razmak, 2,5 cm robovi; važno je, da je obseg od 2700 do 3000 besed), vsebovati mora najmanj tri slike. Slika mora imeti legendo in v besedilu mora biti na ustreznem mestu sklic na sliko.
* Natisnjen seminar oddajte dva tedna pred predstavitvijo vsakemu od recenzentov (docentu ga pošljite po e-pošti v formatu .doc ali .docx).
* Recenzenti do dneva določenega v tabeli določijo popravke in podajo oceno pisnega dela.
* Ustna predstavitev sledi na dan, ki je vpisan v tabeli. Za predstavitev je na voljo 20-30 minut. Recenzenti morajo biti na predstavitvi prisotni.
* Predstavitvi sledi razprava. Recenzenti podajo oceno predstavitve in postavijo najmanj dve vprašanji.
* Na dan predstavitve morate docentu oddati končno (popravljeno) in natisnjeno verzijo seminarja v enem izvodu.
* Seminarska naloga in povzetek morajo biti v slovenskem jeziku!

==Ocenjevanje seminarjev==
Recenzenti ocenijo seminar tako, da izpolnijo [[https://spreadsheets.google.com/viewform?hl=en&formkey=dE1aOFU1aE1iMlBrNEJzLTRGeTdWZXc6MQ#gid=0 recenzentsko poročilo]] na spletu.

== Mnenje o predstavitvi ==
Vsak posameznik '''mora''' oceniti seminar, tako da odda svoje [https://spreadsheets.google.com/viewform?hl=en&formkey=dDlsbDlnclNrc3dIS2otRFdxUEFTNnc6MQ#gid=0 mnenje] najkasneje v treh dneh po predstavitvi. Kdor na seminarju ni bil prisoten, mnenja '''ne sme''' oddati.

==Urejanje spletnih strani na wikiju==
Wiki so razvili zato, da lahko spletne vsebine ureja vsakdo. Ukazi so preprosti, dokler si ne zamislite česa prav posebnega. Vseeno pa je Word v primerjavi z wikijem pravo čudežno orodje... Če imate težave z oblikovanjem besedila, si preberite poglavje o urejanju wiki-strani na Wikipediji ([http://en.wikipedia.org/wiki/Help:Editing tule] v angleščini in [http://sl.wikipedia.org/wiki/Wikipedija:Urejanje_strani tu] v slovenščini). Pomaga tudi, če pogledate, kako je zapisana kakšna stran, ki se vam zdi v redu: kliknite na zavihek 'Uredite stran' in si poglejte, kako so vpisane povezave, kako nov odstavek in podobno. ''Na koncu seveda pod oknom za urejanje kliknite na 'Prekliči'.''

==Citiranje virov==
Citiranje je možno po več shemah, važno je, da se v seminarju držite ene same.
Temeljno načelo je, da je treba vir navesti na tak način, da ga je mogoče nedvoumno poiskati.
Za citate v naravoslovju je najpogostejše citiranje po pravilniku ISO 690. [http://www.zveza-zotks.si/gzm/dokumenti/literatura.html Pravila], ki upoštevajo omenjeni standard, so pripravili pri ZTKS. Sicer pa ima vsaka revija lahko svoj način citiranja, ki ga je treba pri pisanju članka upoštevati. 
'''Citiranje knjig:''' 
Priimek, I. ''Naslov''. Kraj: Založba, letnica. 
Priimek, I. ''Naslov: podnaslov''. Izdaja. Kraj: Založba, letnica. Zbirka, številka. ISBN. 

Boyer, R. ''Temelji biokemije''. Ljubljana: Študentska založba, 2005. 
Glick BR in Pasternak JJ. ''Molecular biotechnology: principles and applications of recombinant DNA''. 3. izdaja. Washington: ASM Press, 2003. ISBN 1-55581-269-4. 
Če so avtorji trije, je beseda in med drugim in tretjim avtorjem. Če so avtorji več kot trije, napišemo samo prvega in dopišemo ''et al''. (in drugi, po latinsko). Vse, kar je latinsko, pišemo poševno (npr. tudi imena rastlin in živali, pojme ''in vivo'', ''in vitro'' ipd.).

'''Citiranje člankov:''' 
Priimek, I. Naslov. ''Naslov revije'', letnica, letnik, številka, strani. 
Lartigue C. ''et al''. Genome transplantation in bacteria: changing one species to another. ''Science'', 2007, letn. 317, str. 632-638.

Alternativni način citiranja (predvsem v družboslovju) je po pravilih APA, kjer članke citirajo takole: 
Priimek, I. (letnica, številka). Naslov. Naslov revije, strani. 
Lartigue C. ''et al.'' (2007, 317) Genome transplantation in bacteria: changing one species to another. ''Science'', 632-638.

Revija Science uporablja skrajšani zapis: 
C. Lartigue ''et al''. Science 317, 632 (2007) 

V diplomah na FKKT je treba navesti vire tako, da izpišete tudi naslov citiranega dela in strani od-do (ne samo začetne).

'''Citiranje spletnih virov:''' 
Priimek, I. ''Naslov dokumenta''. Izdaja. Kraj: Založnik, letnica. Datum zadnjega popravljanja. [Datum citiranja.] spletni naslov 
strangeguitars. ''On the brink of artificial life''. 6. 10. 2007. [citirano 13. 11. 2007] http://www.metafilter.com/65331/On-the-brink-of-artificial-life 
Navedemo čim več podatkov; pogosto vseh iz pravila ne boste našli.

BIO1 Povzetki seminarjev 2011

2011-03-21T15:09:35Z

EvaKnapič:

== Alja Zottel: Vloga imunskega sistema pri nastanku ateroskleroze ==
Glavni vzrok nastanka ateroskleroze je imunski odgovor na lipoproteine majhne gostote oz LDL, ki se kopiči pod endotelom arterijskih žil. Apolipoprotein B100, ki je komponenta LDL, se veže na proteoglikane zunajceličnega matriksa in se pod vplivom različnih radikalov oksidira. OxLDL nato aktivira endotelijske celice, da začnejo proizvajati adhezijske beljakovine, kot sta E-selektin in VCAM-1. Te beljakovine skupaj s kemokini povlečejo monocite, T limfocite in in dendritske celice v endotelijsko plast žile. Monociti se nato pod vplivom M-CSF citokina diferencirajo v makrofage. Makrofagi nato začnejo proizvajati odstranjevalne receptorje. Ti tako lahko prepoznajo oxLDL in ga z endocitozo vsrkajo. Makrofagi se zato napihnejo in spremenijo v »foam cell«. Te celice so najštevilčnejše celice v aterosklerotskih plakih. Dejavniki, ki pospešujejo nastanej ateroskleroze so signalni proteini PRR, T levkociti in proteini CRP. T celice pomagalke izločajo interferon gama, ki privlači monocite. Protein CRP se veže na navadni LDL in tako ga lahko makrofagi, ki imajo receptorje za CRP vsrkajo. Dejavniki, ki preprečujejo nastanek ateroskleroze so B limfociti in protein PPAR. PPAR je receptorski protein oz. transkripcijski faktor, ki preprečuje nastanek »foam cell« celic in vsrkavanje LDL v makrofage. Preprečuje tudi razvoj T celic in povečuje količino HDL v krvi.

== Veronika Jarc: Hepatitis C ==
Hepatitis C(HCV) je nalezljiva bolezen, ki napade ljudi, šimpanze ter nekatere majhne modelne živali. HCV spada med RNA viruse z ovojnico.Razvrščen pa je v rod hepacivirus ter družino flaviviridae. Sestavljen je iz 6 genotipov (1-6), ki se razlikujejo v nukleotidni sekvenci od 30-35%, sedmega pa so odkrili leta 2008 (Gottwein et al., 2008). HCV vsebuje pozitiven trak gena (9,6 kb), ki je sestavljen iz 5´-NCR( non-coding region), 3´- NCR in IRES( internal ribosome entry side). IRES vsebuje odprto bralno ogrodje, ki šifrira strukturne in ne strukturne proteine. Med strukturne proteine spadajo proteinsko jedro, virusna RNA ter dva glikoproteina E1 in E2. Sestavni deli ne strukturnih proteinov pa so hidrofoben protein p7, NS2-3 proteaza, NS3 serin proteaza, NS4A polipeptid, NS4B protein, NS5A protein in NS5B RNA odvisna RNA polimeraza (RdRp).
S pomočjo različnih odkritij, kot so HCVpp(sestavljen iz lipidne ovojnice z E1-E2 proteini, na retrovirusni nukleokapsidi), izoliranje kloniranega gena 2a ter s pomočjo tega gena HCVcc( cell-culture produced HCV), so znanstveniki začeli preučevati življenski cikel in celično strukturo hepatitisa C. To so dosegli z preučevanjem različnih eksperimentalnih modelov kot so imunski odzivi, NK celice in dendritske celice.
Poznamo tudi proteine, ki jih HCv sreča v hepatocitski celici in ti so in tegrin RGE/RGD, LDL receptor, HDL receptor, klaudin okludin in tetraspanin CD81.

== Matja Zalar: Protein p53 ==
Protein p53, včasih imenovan tudi varuh genoma, kodira gen TP53 na sedemnajstem kromosomu. Je eden izmed tako imenovanih tumor-supresorskih proteinov, ki, kot to sporoča že samo ime, zavirajo nastanek in rast tumorjev. Na področju razumevanja delovanja, vloge in strukture proteina p53 in njegovih mutantov se izvaja veliko raziskav. Trenutno je p53 najbolj raziskan tumor-supresorski protein, še zdaleč pa ni edini. Gre za protein, ki se kopiči v jedru in z vezavo na DNA v obliki teramera nadzoruje in regulira procese kot so apoptoza, zaustavitev celičnega cikla in popravljanje poškodovane DNA. Za raziskovalce je še posebno zanimiv zaradi dejstva, da v nemutirani obliki zavira nastanek in rast tumojev, njegove GOF mutirane oblike pa pripomorejo k nenadzorovani delitvi celic in nastanku rakastih tkiv. Veliko raziskav se ukvarjaja z iskanjem snovi, ki bi obnovile osnovno obliko p53, oziroma uničile mutantske oblike p53 v rakastih celicah ter s tem uničile tumor. To pa bi lahko bistveno izboljšalo tehnike zdravljenja rakavih obolenj in odziv človeškega organizma na ta zdravljenja. Odkrili so že kar nekaj takšnih snovi (RITA, PRIMA, nutlin3), ki pa jih še vedno testirajo in še niso v redni uporabi pri zdravljenju rakavih obolenj.

== Andrej Vrankar: Androgena alopecija ==
Na podlagi raziskav, ki so jih znanstveniki izvedli na celičnih vzorcih posameznikov z androgeno alopecijo, so ugotovili, da je bila domneva, da je za nastanek AGA kriv propad matičnih celic v lasnem mešičku oziroma, propad samega lasnega mešička napačna. Raziskave so pokazale ravno nasprotno in sicer, da se matične celice tudi v plešastem lasišču posameznika z AGA ohranjajo in da lasni mešički ne propadejo, vendar se le zelo skrčijo. So pa ugotovili, da se število celic imenovanih predniške celice v plešastem lasišču močno zmanjša, kar je eden od glavnih vzrokov za nastanek AGA, saj so prav predniške celice tiste, ki so zaslužene za rast las. Čeprav se dednost smatra kot glavni vzrok za nastanek AGA, pa tudi hormoni igrajo pomembno vlogo. Pri moških je to moški hormon testosteron, ki se s pomočjo encima 5-α-reduktaze v lasno mešičnih celicah pretvarja v svojo bolj aktivno obliko dihidrotestosteron (DHT). Ta se se nato s posebno vezjo veže na androgene receptorje v lasnih mešičkih, kar sproži posebne procese, ki skrajšajo anageno fazo celičnega cikla. Zaradi skrajšanja te faze las prej prestopi v telogeno fazo in izpade. Kako občutljivi so lasni mešički na androgene pa je seveda gensko pogojeno.

== Sandi Botonjić: Tioredoksinu podoben protein (TXNL2) ščiti kancerogene celice pred oksidativnim stresom ==
Kisikovi radikali, ki povzročajo oksidativni stres lahko v skrajnem primeru poškodujejo DNA in tako povzročijo nenadzorovano delitev celic, kar pomeni nastanek raka v organizmu. Hkrati pa je raven kisikovih radikalov v rakastih celicah višja, kot v zdravih, in sicer zaradi onkogenih stimulacij, povečane presnovne aktivnosti ter okvare mitohondrijev. Toda rakave celice imajo, kot protiutež tudi močan antioksidantni mehanizem s katerim zavirajo programirano celično smrt.

Raziskovalci so tekom analiziranja večih tkiv, ki so obolela z različnimi vrstami raka ugotovili, da je pri vseh povečana raven [http://www.thesgc.org/structures/structure_images/2WZ9_400x400.png tioredoksinu podobnega proteina - TXNL2]. Zatem so izvajali poskuse na miših tako, da so jim vbrizgali kancerogene eritrocite in ko so se pojavili simptomi tumorja – so jim vbrizgali še protein TXNL2. Ugotovili so, da protein TXNL2 zavira rast rakavih celic. Proučevali so tudi vpliv proteina TXNL2 v mišjih zarodkih. Prišli so do zaključka, da protein TXNL2 regulira raven kisikovih radikalov tako pri živečih organizmih, kot med embriogenezo.

Znanstveniki so prepričani, da je protein TXNL2 potencialna tarča bioloških zdravil v prihodnosti. Namreč monoklonska protitelesa (med katere spade tudi TXNL2) za zdravljenje raka z vezavo na receptorje za rastne dejavnike blokirajo celično rast in diferenciacijo ter tako zaustavijo rast tumorja. Zaustavijo lahko tudi rast tumorskega ožilja in s tem posredno onemogočajo rast tumorjev in metastaziranje. Med mehanizme delovanja monoklonskih protiteles, spada tudi ciljanje drugih efektorskih molekul na mesta delovanja - kot so npr. kisikovi radikali. Raziskave so potrdile, da to velja tudi za protein TXNL2.

== Ana Dolinar: Prilagojena ali prilagodljiva imunost? Primer naravnih celic ubijalk ==
Naravne celice ubijalke (NK celice) so vrsta levkocitov. V človeškem telesu so zadolžene za uničevanje patogenih organizmov s pomočjo za celice strupenih snovi. Na površini imajo pet skupin receptorjev: aktivacijske, inhibitorne, kemotaksične in citokine ter adhezijske receptorje.

Njihova aktivacija je odvisna od vezave ligandov na površinske receptorje NK celice. Če je vezanih več inhibitornih ligandov kot aktivacijskih, potem se NK celica ne aktivira, ker inhibitorni ligandi zavrejo delovanje NK celice. V primeru, da se veže več aktivacijskih kot inhibitornih ligandov ali pa se slednji sploh ne vežejo, se NK celica aktivira ([http://www.georg-speyer-haus.de/agkoch/research/subframe_en.htm aktivirana NK celica-rumeno, tarčna celica-rdeče]). Vezava kemotaksičnih ligandov vpliva na gibanje molekule zaradi kemičnih signalov, vezava citokinov spodbuja rast celic ali sintezo snovi, ki jih potrebuje imunski sistem, vezava adhezijskih ligandov pa omogoča pritrjanje NK celice na tarčno celico.

Raziskovalci se trudijo, da bi našli optimalno imunoterapijo, pri kateri bi sodelovale NK celice. Te terapije bi bile uporabne predvsem pri rakavih obolenjih, vendar so možnosti tudi pri obolenjih z virusom HIV ali z virusom hepatitisa C. Ta način imunoterapije je mogoč, ker večina tumorskih celic in virusov ne izraža MHC tipa 1, pomembnega inhibitorskega liganda za NK celice. [http://media.wiley.com/CurrentProtocols/IM/ima01n/ima01n-fig-0004-1-full.gif Zgradba MHC-1 molekule, prikazana z Ribbonovim diagramom in vezanim peptidom (A) ter površinska struktura molekule z vezanim peptidom (C). Slika B prikazuje molekulo MHC-2 z vezanim peptidom.]

== Urška Rauter: Razvojne vloge Srf, kortikalnega citoskeleta in celične oblike pri orientaciji epidermalnega vretena ==
Mehanizem nastajanja polariziranega epidermalnega sloja, ki s procesoma stratifikacije in diferenciacije tvori kožo, regulira več različnih med seboj v komplekse povezanih bioloških molekul. Trije najbolj osnovni procesi so delovanje proteinov aktina, orientacija vretena in sistem celične signalizacije. Znanstveniki pa so v obširni raziskavi potrdili tudi pomembno vlogo t. i. Srf proteina (serum response factor protein), transkripcijskega dejavnika, katerega pomembna vloga je regulacija celične diferenciacije.

Srf je transkripcijski dejavnik, ki se veže na določen, njemu ustrezen receptorski element; Sre (serum response element), to so predvsem geni v zgodnjem razvoju, geni za razvoj nevronov in mišična gena (proteina) aktin in miozin. Ker je njegova primarna funkcija regulacija ekspresije naštetih genov, odločilno vpliva na celično rast in diferenciacijo, prenos med nevroni in razvoj mišic.

Namen raziskave je obširen. Rezultati obetajoči. Dokazali so pomembno vlogo Srf proteina pri marsikaterem mehanizmu/procesu v embrionalnem razvoju. Tako recimo Srf odločilno vpliva na diferenciacijo celic, saj izguba le-tega povzroči kaotično deljenje in diferenciacijo celic med več plastmi epidermisa. Nadalje vpliva tudi na pravilno vzpostavitev polarnosti bazalne lamine in še najbolj ključno na tvorbo aktinsko-miozinskega skeleta, ki je nujen za pravilno mitozo, posledično za obliko in trdnost celice. Orientacija vretena in asimetrično dedovanje sta po zadnjih raziskavah osrednja mehanizma, ki omogočata matičnim celicam samostojno obnovi in diferenciacijo v pravilni smeri. Rezultati kažejo, da lahko takšne signale pošiljamo preko Srf proteina in aktinsko-miozinskega skeleta, za pravilno tvorbo in nadzirano regulacijo orientacije vretena, asimetrične celične delitve in nasploh usodo posamezne celice. Rezultati razkrivajo nove pojasnitve bioloških procesov, ki sodelujejo pri tvorbi morfologije epidermisa.

== Špela Pohleven: Prioni ==

Prioni so patogeni proteini, ki se od svojih nepatogenih, normalnih, v zaporedju aminokislin enakih dvojnikov, razlikujejo v 3D strukturi – imajo večji del β ploskev. Poznamo več vrst prionov, toda običajno govorimo le o proteinu PrP, ki je prisoten pri ljudeh in živalih. Ostali so namreč značilni za glive, ki so tako primerne za razne raziskave.
Za prione je značilno povezovanje v nitaste polimere, ki jih imenjujemo amiloidi. Znanstveniki domnevajo, da je prav njihova urejena struktura tista, zaradi katere so slabo topni v detergentih in odporni na proteaze.
Najbolj nenavadna lastnost prionov pa je njihova zmožnost širjenja brez potrebe po DNA in RNA. V zvezi s tem potekajo številne raziskave, saj prioni povzročajo številne smrtne bolezni, kot so Creutzfeldt-Jakobova bolezen, smrtonosna družinska nespečnost in druge. Z informacijami, ki jih tako pridobivajo, je možnost za odkritje zdravila večja.
Pri eni od nedavnih raziskav so tako ugotovili, da obstajata dve prionski obliki proteina PrP – infektivna in toksična. Za raziskave so uporabili miši z različnim izražanjem gena PRNP za PrP protein. Vse so okužili s prioni praskavca (ena od prionskih bolezni). Vse so dosegle enak prag infektivnosti, toda smrt ni nastopila istočasno. Iz meritev so znanstveniki prišli do zaključka, da morata obstajati dve različni obliki. To pa je le izhodišče za nove raziskave.

== Maša Mohar: Sladkorna bolezen, kot bolezen imunskega sistema ==

Diabetes mellitus je kronična motnja metabolizma beljakovin, lipidov in ogljikovih hidratov. Nastane zaradi zmanjšane funkcije proizvajanja insulina v telesu. Njen vzrok pa je lahko studi zmanjšana sposobnost telesnih celic za pravilno izkoriščanje insulina. Tip 2 je od insulina neodvisen diabetes (NIDDM). Ta tip ima 80-90% vseh pacientov in se pojavi v odraslem obdobju življenja, spodbudijo ga lahko različni mehanizmi, in za nekatere se še ne ve točno kako pride do tega, je pa res da k temu veliko pripomore nezdrav način življenja in seveda dednost. Prav tako se diabetes tipa 2 deli v dve skupini in sicer na debeli tip, ki ga ima približno 80% vse populacije in na ne debeli tip.
Da je T2D bolezen imunskega sistema pa ugotovimo s tem ko vidimo kako se telo odzovena določene mehanizme, ki sprožijo to bolezen. To so oksidativni stres, stres ER( endoplazemski retikel), lipotoksičnost in glukotoksičnost. Prav tako je potrebno poudariti, da ima diabetes tipa 2 svoje metabolne karakteristike in skupaj s temi patogenimi mehanizmi tvori formulo za nastanek bolezni. Seveda lahko pri T2D pride tudi do dolgoročnih komplikacij, kot so makro in mikro- vaskularne bolezni, problemi z ledvicami, očmi in živci. Te pa so glavni dejavniki za povzročitev hujšega bolezenskega stanja in ne nazadnje tudi smrti zaradi diabetesa.

== Mirjam Kmetič: Regulacija celičnega metabolizma železa ==

Železo je pomemben mikroelement, ki ga vezanega na proteine, vsebujejo skoraj vsa živa bitja. Celice sesalcev potrebujejo zadostno količino železa, da zadovoljijo metabolne potrebe ali dosežejo specializirane funkcije. Vsekakor pa je železo potencialno strupeno, še posebej v obliki Fe2+ ionov, ki katalizirajo pretvorbo vodikovega peroksida v proste radikale, ti pa poškodujejo veliko celičnih struktur (DNA, proteine, lipide...) in posledično celica lahko celo odmre. Vse oblike življenja se temu izognejo tako, da vežejo železove ione na proteine in tako hkrati izkoristijo njegove ugodnosti. Železo se prenaša v tkivo ob pomoči kroženja transferina, prenašalca, ki veže železo v plazmi, katerega predvsem sproščajo črevesne resice in retikuloendotelni makrofagi. Z železom bogat transferin se veže na membranski transferin receptor 1, kar se odraža z endocitozo in sprejemom te kovine. Sprejeto železo se prenese do mitohondrija za sintezo hema ali železo-žveplovih proteinov, ki so bistveni deli mnogih metaloproteinov. Presežno železo se skladišči in detoksificira v feritinu, ki je v citosolu. Metabolizem železa je nadzorovan na različnih nivojih in z raznovrstnimi mehanizmi. Pri uravnavanju je zelo pomemben sistem IRE (iron-responsive element)/IRP (iron-regulatory protein), dobro poznano post-transkripcijsko regulatorno vezje, ki ne le vzdržuje homeostazo v različnih tipih celic, ampak tudi prispeva k sistemskemu ravnovesju železa.

== Lea Kepic: Agonisti adrenoreceptorjev β2 ==

Vloga receptorjev v organizmih je zelo pomembna saj prenaša vse potrebne informacije za delovanje. Delimo jih na ionotropne in metabotropne. Največja skupina metabotropnih receptorjev pripada receptorjem, ki so sklopljeni s proteinom G. Mednje spadajo tudi adrenergični receptorji ali adrenoreceptorji. Adrenoreceptorji so tarčni za katekolamine (fight or flight hormoni) med katere spadajo adrenalin, noradrenalin in dopamin. V svojem seminarju sem se posvetila predvsem podskupini β2 (β2-AR) in njihovim agonistom. Agonisti so spojine, ki se selektivno vežejo na specifične receptorje, ki sprožijo nadaljnji odziv. Njegova naloga je posnemanje naravno obstoječih (endogenih) molekul, kot so na primer hormoni. Najbolj pogost in učinkovit agonist za β2-AR je izoprenalin, med hormoni pa je najboljši adrenalin. S pomočjo eksperimentov znanstveniki raziskujejo posebnosti v zgradbi predvsem kristalnih struktur, tvorbo vezi z različnimi spojinami, konformacijske spremembe, vpliv inhibitorjev, ravnotežna stanja ter energijska pretvarjanja. Rezultati teh raziskav so izhodišče za praktično uporabnost agonistov. Zaradi njihovih lastnosti jih vedno več uporabljamo v medicini za zdravnjenje plujčnih bolezni; predvsem astme in bronhitisa. To področje za enkrat še ni do dobra raziskano zato jih navadno uporabljamo le kot dodatke drugim zdravilom. Raziskani pa so že tudi nekateri negativni učinki na telo.

== Iza Ogris: Zakaj imajo možgani glikogen? ==

Glikogen se v možganih nahaja v precej manjših koncentracijah kot v jetrih in mišicah.Pojavi se vprašanje o njegovi vlogi v možganih in kje se nahaja. Glikogen vsebujejo astrocite- glia celice, ki obdajajo nevrone in skbijo za koncentracijo ionov v izvenceličnem prostoru ter dovajanje določenih snovi nevronom. Ko se med aktivnostjo nevronov v izvenceličnem prostoru kopičijo kalijevi ioni, jih astrocite začnejo privzemati z K/Na ATPazo. Posledično se v astrocitah zviša nivo AMP, kar stimulira delovanje encima glikogen fosforilaze (razgradnja glikogena). Astrocite med nevronsko aktivnostjo privzemajo tudi živnčni prenašalec glutamat iz sinaps, ki tudi posredno povzroča padec energije v astrocitah. Ko se nivo glukoze v dejavnih nevronih znižuje, se medtem v astrocitih povečuje. Koncentracija glukoze je nato v astrocitih večja kot v izvencelični tekočini in nevronih, zato se ustvari koncentracijski gradient kar omogoči pot glukoze iz astrocitov v nevrone. Pri vzdrževanju glukoze se tako razgradnja glikogena izkaže za bolj učinkovito kot le privzem glukoze iz krvi. Razkriva se izvor in usoda glukozne rezerve.

== Ines Kerin: Kanabinoidi za zdravljenje shizofrenije? Uravnotežena nevrokemična sestava za škodljive in terapevtske učinke uživanja konoplje ==

Že desetletja velja prepričanje, da je uživanje konoplje eden pomembnih dejavnikov za nastanek in razvoj shizofrenije. Vendar so v novejših raziskavah odkrili, da naj bi kanabinoidi, psihoaktivne substance v konoplji, izboljšali nevropsihološke učinke in negativne simptome, ter imeli antipsihotične lastnosti pri ljudeh s shizofrenijo. Shizofrenija je huda duševna bolezen iz skupine psihoz. Simptome shizofrenije povzroča spremenjena količina določenih snovi v možganih, in sicer živčnih prenašalcev, ki omogočajo medsebojno komunikacijo možganskih celic. Motnje v komunikaciji pa povzročajo spremembo v delovanju možganov. Pomembno vlogo ima pri bolezni dopamin, ki lahko s prevelikim sproščanjem izzove nekatere simptome.
Shizofrenijo zdravijo s pomočjo antipsihotikov, ki imajo podobne lastnosti kot kanabinoidi v konoplji. Vendar se učinki konoplje od učinkov antipsihotikov nekoliko razlikujejo. Pri negativnih simptomih konoplja, tako kot antipsihotiki, spodbuja sproščanje in delovanje dopamina. Manj znano pa je, ali zavira ali spodbuja delovanje ostalih petih nevrotransmiterjev (serotonina, acetilholina, noradrenalina, glutamina in GABA). Na pozitivne simptome ima konoplja, kot je vidno v tabeli lahko tako koristne kot nekosristne učinke. Simptome lahko izboljša z zaviranjem sproščanja serotonina, acetilholina in noradrenalina. V primeru dopamina, glutamata in GABA ima konopla negative učinke, saj v nasprotju z antipsihotiki, poveča sproščanje dopamina in zavira delovanje glutamata in GABA. Obstajajo dokazi, da imajo kanabinoidi zdravilne učinke na pozitivne in negativne simptome pri shizofreniji. Vendar to poglavje še ni zaključeno in se izvajajo še nadalnje raziskave v tej smeri.

== Eva Knapič: Kako virusi vodijo delovanje celice. ==
Virusi so geni obdani z zaščitno proteinsko ovojnico. Za izražanje teh genov, da lahko naredijo proteine in podvojijo kromosome, je potrebno, da vstopijo v celico in uporabijo celične mehanizme, saj sami tega niso zmožni. Poznamo več vrst virusov. Posebnost evkariontskih virusov je sposobnost posnemanja kratkih linearnih motivov proteinov poznanih pod kratico SLiMs. To so deli proteinov, ki so odgovorni za posredovanje med nekaterimi celičnimi funkcijami. So zelo kratki, večinoma nekje od 3 do 10 aminokislin. Motivi sodelujejo pri vezavi proteinih, pri prepoznavanju post-translacijske modifikacije encimov, pri usmerjanju proteinov v celične razdelke in pa so prisotni na cepitvenih mestih proteina. S posnemanjem različnih motivov lahko virusi prevzamejo nadzor nad celico. Najpogostejši mehanizmi prevzema nadzora so uporaba celičnega transporta, manipuliranje signalnega transporta, nadzor proteinov v celici, regulacija prepisovanja, sprememba modifikacije gostiteljevega proteina in usmerjanje modifikacije proteinov.
Uporaba proteinskih motivov v celici in lahko posnemanje le teh predstavlja šibkost v celični organiziranosti, saj virusi s pridom izkoriščajo to v svojo korist. Posnemanje motivov virusom omogoča, da sami vodijo delovanje celice in se sami s pomočjo celičnih mehanizmov enostavno razmnožujejo in tako hitro okužijo celoten organizem.
V nadalje bodo potekale raziskave za izkoriščanje posnemanja motivov v namene zdravljenja virusnih okužb.

BIO1-seminar 2011

2011-03-10T18:31:25Z

EvaKnapič: /* Seznam seminarjev */

= Temelji biokemije- seminar =

Seminarje vodi doc. dr. Gregor Gunčar in so na urniku vsak ponedeljek od 10:00 do 11:30.

Ocena seminarjev predstavlja ??% končne ocene in vsebuje vse točke, ki jih študent/ka lahko zbere pri seminarju in ostalih dejavnostih, ki niso del pisnega izpita.

== Seznam seminarjev ==
{| border="1" cellpadding="4" cellspacing="0" style="border:#c9c9c9 1px solid; margin: 1em 1em 1em 0; border-collapse: collapse;"
| align="center" style="background:#f0f0f0;"|'''Ime in priimek'''
| align="center" style="background:#f0f0f0;"|'''Slovenski naslov članka'''
| align="center" style="background:#f0f0f0;"|'''Faktor vpliva revije'''
| align="center" style="background:#f0f0f0;"|'''Rok za oddajo'''
| align="center" style="background:#f0f0f0;"|'''Rok za recenzijo'''
| align="center" style="background:#f0f0f0;"|'''Datum predstavitve'''
| align="center" style="background:#f0f0f0;"|'''Recenzent 1'''
| align="center" style="background:#f0f0f0;"|'''Recenzent 2'''
| align="center" style="background:#f0f0f0;"|'''Recenzent 3'''
|-
| BOTONJIĆ SANDI||[http://wiki.fkkt.uni-lj.si/index.php/BIO1_Povzetki_seminarjev#Sandi_Botonji.C4.87:_Tioredoksinu_podoben_protein_.28TXNL2.29_.C5.A1.C4.8Diti_kancerogene_celice_pred_oksidativnim_stresom Tioredoksinu podoben protein (TXNL2) ščiti kancerogene celice pred oksidativnim stresom]
||15.387||28.02.||03.03.||07.03.||RODE URŠKA||KERIN INES||OGRIS IZA
|-
| VRANKAR ANDREJ||Število lasno-mešičnih matičnih celic se v plešastem lasišču moških z androgeno alopecijo ohranja za razliko od števila CD200-rich in CD34-positive lasno-mešičnih predniških celic||||28.02.||03.03.||07.03.||HROVAT KARMEN||BOHNEC IVO||JAVORŠEK KAJA
|-
| ZALAR MATJA||Protein p53||||28.02.||03.03.||07.03.||OGRIS IZA||CRČEK MITJA||ZOTTEL ALJA
|-
| ZOTTEL ALJA||Vloga imunskega sistema pri aterosklerozi||31.434||07.03.||10.03.||14.03.||RADOJKOVIĆ MARKO||KERT DOMINIK||HROVAT KARMEN
|-
| DOLINAR ANA||[http://wiki.fkkt.uni-lj.si/index.php/BIO1_Povzetki_seminarjev#Ana_Dolinar:_Prilagojena_ali_prilagodljiva_imunost.3F_Primer_naravnih_celic_ubijalk Prirojena ali prilagodljiva imunost? Primer naravnih celic ubijalk]||28||07.03.||10.03.||14.03.||RAUTER URŠKA||MOHAR MAŠA||VERBANČIČ JANA
|-
| RAUTER URŠKA||Razvojna vloga Srf, kortikalnega citoskeleta in celične oblike v orientaciji epidermalnega vretena||19.527||07.03.||10.03.||14.03.||MUSTAR JERNEJ||JAVORŠEK KAJA||MOHAR MAŠA
|-
| MOHAR MAŠA||Sladkorna bolezen tipa 2 kot bolezen imunskega sistema||30,006||14.03.||17.03.||21.03.||VENE ROK||RAUTER URŠKA||GORIČAN TJAŠA
|-
| POHLEVEN ŠPELA||Prioni||34||14.03.||17.03.||21.03.||KEPIC LEA||RADOJKOVIĆ MARKO||DOLINAR ANA
|-
| KEPIC LEA||Agonisti adrenoreceptorjev β2||34.48||14.03.||17.03.||21.03.||VRANKAR ANDREJ||BRATOVŠ ANDREJA||MUSTAR JERNEJ
|-
| KMETIČ MIRJAM||Celična regulacija metabolizma železa||5,371||14.03.||17.03.||21.03.||MARIĆ TAMARA||REMŠKAR MAJA||KOMAN KATRA
|-
| JARC VERONIKA||Hepatitis C||3.26||14.03.||21.03.||28.03.||REMŠKAR MAJA||MUSTAR JERNEJ||KEPIC LEA
|-
| KOMAN KATRA||naslov||||14.03.||21.03.||28.03.||ČUPOVIĆ VANA||KARNER TAJA||KMETIČ MIRJAM
|-
| OGRIS IZA||Zakaj imajo možgani glikogen?||||14.03.||21.03.||28.03.||KNAPIČ EVA||BRGLEZ ŽIVA||VRANKAR ANDREJ
|-
| KERIN INES||naslov||||14.03.||21.03.||28.03.||ŠTOK ULA||ŠTEMBERGER ROK||KERT DOMINIK
|-
| VERBANČIČ JANA||naslov||||21.03.||28.03.||04.04.||KARNER TAJA||ZOTTEL ALJA||KNAPIČ EVA
|-
| KNAPIČ EVA||Kako virusi vodijo delovanje celice.||14.101||21.03.||28.03.||04.04.||ZALAR MATJA||POHLEVEN ŠPELA||LORBEK SARA
|-
| REMŽGAR ANA||naslov||||21.03.||28.03.||04.04.||BOTONJIĆ SANDI||LORBEK SARA||ČUPOVIĆ VANA
|-
| GRDADOLNIK MAJA||naslov||||21.03.||28.03.||04.04.||MOHAR MAŠA||REMŽGAR ANA||FRANKO NIK
|-
| JAVORŠEK KAJA||Potencial matične celice pri Parkinsonovi bolezni in molekularni faktorji za tvorbo dopaminergičnih nevronov||4.139||28.03.||04.04.||11.04.||GEC KARMEN||MARIĆ TAMARA||RADOJKOVIĆ MARKO
|-
| BRATOVŠ ANDREJA||Vloga GPCR v patologiji Alzheimerjeve bolezni||26||28.03.||04.04.||11.04.||ZOTTEL ALJA||ČUPOVIĆ VANA||GRDADOLNIK MAJA
|-
| CRČEK MITJA||naslov||||28.03.||04.04.||11.04.||BOHNEC IVO||KMETIČ MIRJAM||BRATOVŠ ANDREJA
|-
| MARIĆ TAMARA||ciljanje kemokinskih receptorjev ob alergijskih obolenjih||5.155||28.03.||04.04.||11.04.||NAVODNIK URŠKA||GEC KARMEN||REMŠKAR MAJA
|-
| ŠTEMBERGER ROK||naslov||||04.04.||11.04.||18.04.||JAVORŠEK KAJA||VRANKAR ANDREJ||BOTONJIĆ SANDI
|-
| LORBEK SARA||naslov||||04.04.||11.04.||18.04.||POHLEVEN ŠPELA||KNAPIČ EVA||VENE ROK
|-
| REMŠKAR MAJA||naslov||||04.04.||11.04.||18.04.||KERIN INES||POVŠE KATJA||CRČEK MITJA
|-
| ČUPOVIĆ VANA||naslov||||04.04.||11.04.||18.04.||REMŽGAR ANA||VERBANČIČ JANA||RODE URŠKA
|-
| RODE URŠKA||naslov||||24.04.||03.05.||09.05.||GRDADOLNIK MAJA||FRANKO NIK||MARIĆ TAMARA
|-
| RADOJKOVIĆ MARKO||naslov||||24.04.||03.05.||09.05.||FRANKO NIK||VENE ROK||POVŠE KATJA
|-
| VENE ROK||naslov||||24.04.||03.05.||09.05.||VERBANČIČ JANA||NAVODNIK URŠKA||ZALAR MATJA
|-
| FRANKO NIK||naslov||||24.04.||03.05.||09.05.||ŠTEMBERGER ROK||HROVAT KARMEN||BOHNEC IVO
|-
| HROVAT KARMEN||naslov||||04.05.||09.05.||16.05.||KERT DOMINIK||JARC VERONIKA||KARNER TAJA
|-
| AMBROŽIČ MATEVŽ||naslov||||04.05.||09.05.||16.05.||LORBEK SARA||KEPIC LEA||REMŽGAR ANA
|-
| NAVODNIK URŠKA||naslov||||04.05.||09.05.||16.05.||AMBROŽIČ MATEVŽ||ŠTOK ULA||ŠTEMBERGER ROK
|-
| BRGLEZ ŽIVA||naslov||||09.05.||16.05.||23.05.||DOLINAR ANA||BOTONJIĆ SANDI||JARC VERONIKA
|-
| KARNER TAJA||naslov||||09.05.||16.05.||23.05.||KOMAN KATRA||OGRIS IZA||NAVODNIK URŠKA
|-
| KERT DOMINIK||naslov||||09.05.||16.05.||23.05.||GORIČAN TJAŠA||GRDADOLNIK MAJA||RAUTER URŠKA
|-
| MUSTAR JERNEJ||naslov||||16.05.||23.05.||30.05.||JARC VERONIKA||AMBROŽIČ MATEVŽ||BRGLEZ ŽIVA
|-
| GEC KARMEN||naslov||||16.05.||23.05.||30.05.||POVŠE KATJA||ZALAR MATJA||AMBROŽIČ MATEVŽ
|-
| GORIČAN TJAŠA||naslov||||16.05.||23.05.||30.05.||KMETIČ MIRJAM||RODE URŠKA||POHLEVEN ŠPELA
|-
| BOHNEC IVO||naslov||||23.05.||30.05.||06.06.||CRČEK MITJA||GORIČAN TJAŠA||ŠTOK ULA
|-
| ŠTOK ULA||naslov||||23.05.||30.05.||06.06.||BRGLEZ ŽIVA||DOLINAR ANA||KERIN INES
|-
| nihce ||naslov||||23.05.||30.05.||06.06.||BRATOVŠ ANDREJA||KOMAN KATRA||GEC KARMEN
|}

==Naloga==
* samostojno pripraviti seminar, katerega osnova je znanstveni članek s področja biokemije, ki ga po želji izberete v reviji s področja biokemije, ki ima faktor vpliva večji kot 3 in je bil objavljen v letu 2011. Poleg tega članka morate za seminar uporabiti še najmanj pet drugih virov! http://www.cobiss.si/scripts/cobiss?command=CONNECT&base=JCR
* osnovni članek in naslov pošljete meni, najkasneje pet dni pred rokom za oddajo (rok-5), da ocenim, če je primeren za predstavitev. Naslov vpišete v tabelo, takoj ko ste si ga izbrali!
* [[BIO1 Povzetki seminarjev|Povzetek seminarja]] opišete na wikiju v približno 200 besedah - najkasneje do dne ko morate oddati seminar recenzentom. Povezave do slik so dobrodošle, niso pa nujne.
* Povezavo do povzetka vnesete v tabelo seminarjev tekočega letnika.
* Seminar pripravite v obliki seminarske naloge (pisava 12, enojni razmak, 2,5 cm robovi; važno je, da je obseg od 1800 do 2000 besed), vsebovati mora najmanj eno sliko. Slika mora imeti legendo in v besedilu mora biti na ustreznem mestu sklic na sliko.
* Natisnjen seminar oddajte do roka vsakemu od recenzentov (docentu ga pošljite po e-pošti v formatu .doc ali .docx).
* Recenzenti do dneva določenega v tabeli določijo popravke in podajo oceno pisnega dela, v predpisanem formatu elektronskega obrazca na internetu.
* Ustna predstavitev sledi na dan, ki je vpisan v tabeli. Za predstavitev je na voljo 15 minut. Recenzenti morajo biti na predstavitvi prisotni.
* Predstavitvi sledi razprava- 5 minut. Recenzenti podajo oceno predstavitve in postavijo vsak vsaj dve vprašanji.
* Na dan predstavitve morate docentu oddati končno (popravljeno) in natisnjeno verzijo seminarja v enem izvodu.
* Seminarska naloga in povzetek na wikiju morajo biti v slovenskem jeziku!

==Ocenjevanje seminarjev==
Recenzenti ocenijo seminar tako, da izpolnijo [[https://spreadsheets.google.com/viewform?formkey=dFM2SktfM3Q4VU1wNUQzdU45OTlWVXc6MA recenzentsko poročilo]] na spletu.

== Mnenje o predstavitvi ==
Vsak posameznik '''mora''' oceniti seminar, tako da odda svoje [https://spreadsheets.google.com/viewform?formkey=dFd3TGhLV3ZSa2xsLVlmMVVUaEFURWc6MA mnenje] najkasneje v treh dneh po predstavitvi. Kdor na seminarju ni bil prisoten, mnenja '''ne sme''' oddati.

==Urejanje spletnih strani na wikiju==
Wiki so razvili zato, da lahko spletne vsebine ureja vsakdo. Ukazi so preprosti, dokler si ne zamislite česa prav posebnega. Vseeno pa je Word v primerjavi z wikijem pravo čudežno orodje... Če imate težave z oblikovanjem besedila, si preberite poglavje o urejanju wiki-strani na Wikipediji ([http://en.wikipedia.org/wiki/Help:Editing tule] v angleščini in [http://sl.wikipedia.org/wiki/Wikipedija:Urejanje_strani tu] v slovenščini). Pomaga tudi, če pogledate, kako je zapisana kakšna stran, ki se vam zdi v redu: kliknite na zavihek 'Uredite stran' in si poglejte, kako so vpisane povezave, kako nov odstavek in podobno. ''Na koncu seveda pod oknom za urejanje kliknite na 'Prekliči'.''

==Faktor vpliva==
Faktor vpliva (angl. impact factor) neke revije pove, kolikokrat so bili v poprečju citirani članki v tej reviji v dveh letih skupaj pred objavo tega faktorja. Faktorje vpliva za posamezno revijo lahko najdete v [http://www.cobiss.si/scripts/cobiss?command=CONNECT&base=JCR COBISS-u]. V polje "Naslov revije" vnesite ime revije za katero želite izvedeti faktor vpliva in pritisnite na gumb POIŠČI. V skrajnem desnem stolpcu se bodo izpisali faktorji vpliva za revije, ki ustrezajo vašim iskalnim kriterijem. Zadetkov za posamezno revijo je več zato, ker so navedeni faktorji vpliva za posamezno leto. Za leto 2011 faktorji vpliva še niso objavljeni, zato se orientirajte po faktorjih vpliva zadnjih par let. Če faktorja vpliva za vašo izbrano revijo ne najdete v bazi COBISS, potem izberite članek iz kakšne druge revije.

==Citiranje virov==
Citiranje je možno po več shemah, važno je, da se v seminarju držite ene same.
Temeljno načelo je, da je treba vir navesti na tak način, da ga je mogoče nedvoumno poiskati.
Za citate v naravoslovju je najpogostejše citiranje po pravilniku ISO 690. [http://www.google.com/url?sa=t&source=web&cd=6&sqi=2&ved=0CEUQFjAF&url=http%3A%2F%2Fwww.tre.sik.si%2Fmain%2Fpomoc%2Ffiles%2Fcitiranje_in_navajanje_virov.pdf&rct=j&q=citiranje%20po%20pravilniku%20ISO%20690&ei=jPBqTe6FC9DKswaWk-TmDA&usg=AFQjCNF8r6X9Y781sanDObaXNdCew4suUg&sig2=cTqKObSJsTicekWGRGa72g&cad=rja Pravila], ki upoštevajo omenjeni standard, so pripravili pri ZTKS. Sicer pa ima vsaka revija lahko svoj način citiranja, ki ga je treba pri pisanju članka upoštevati. 
'''Citiranje knjig:''' 
Priimek, I. ''Naslov''. Kraj: Založba, letnica. 
Priimek, I. ''Naslov: podnaslov''. Izdaja. Kraj: Založba, letnica. Zbirka, številka. ISBN. 

Boyer, R. ''Temelji biokemije''. Ljubljana: Študentska založba, 2005. 
Glick BR in Pasternak JJ. ''Molecular biotechnology: principles and applications of recombinant DNA''. 3. izdaja. Washington: ASM Press, 2003. ISBN 1-55581-269-4. 
Če so avtorji trije, je beseda in med drugim in tretjim avtorjem. Če so avtorji več kot trije, napišemo samo prvega in dopišemo ''et al''. (in drugi, po latinsko). Vse, kar je latinsko, pišemo poševno (npr. tudi imena rastlin in živali, pojme ''in vivo'', ''in vitro'' ipd.).

'''Citiranje člankov:''' 
Priimek, I. Naslov. ''Naslov revije'', letnica, letnik, številka, strani. 
Lartigue C. ''et al''. Genome transplantation in bacteria: changing one species to another. ''Science'', 2007, letn. 317, str. 632-638.

Alternativni način citiranja (predvsem v družboslovju) je po pravilih APA, kjer članke citirajo takole: 
Priimek, I. (letnica, številka). Naslov. Naslov revije, strani. 
Lartigue C. ''et al.'' (2007, 317) Genome transplantation in bacteria: changing one species to another. ''Science'', 632-638.

Revija Science uporablja skrajšani zapis: 
C. Lartigue ''et al''. Science 317, 632 (2007) 

V diplomah na FKKT je treba navesti vire tako, da izpišete tudi naslov citiranega dela in strani od-do (ne samo začetne).

'''Citiranje spletnih virov:''' 
Priimek, I. ''Naslov dokumenta''. Izdaja. Kraj: Založnik, letnica. Datum zadnjega popravljanja. [Datum citiranja.] spletni naslov 
strangeguitars. ''On the brink of artificial life''. 6. 10. 2007. [citirano 13. 11. 2007] http://www.metafilter.com/65331/On-the-brink-of-artificial-life 
Navedemo čim več podatkov; pogosto vseh iz pravila ne boste našli.

BIO1-seminar 2011

2011-02-22T14:12:59Z

EvaKnapič: /* Seznam seminarjev */