Wiki FKKT - User contributions [en]

2015-bionano-seminar

2015-05-03T22:04:28Z

Katarina Uršič:

= 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||Avtomobilski encimski katalizator||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||Test občutljivosti na gluten z neprebavljivo kapsulo||31.03.||02.04.||Varja Božič||Klara Tereza Novoselc
|-
| Katja Lovrin||Mitja Crček||Uporaba nitrifikacijskih encimov pri kmetovanju||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||Antimaček®||12.04.||14.04.||Katja Lovrin||Veronika Jarc
|-
| Vida Špindler||Jernej Pušnik||||12.04.||14.04.||Saša Balažic||Iza Ogris
|-
| Jasmina Sedmak||Maxi Sagmeister||Brezžični zobni nanobiosenzor ||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||Biorazgradljivi žvečilni gumi z antibakterijskimi lastnostmi||03.05.||05.05.||Simon Preložnik||Kim Kos
|-
| Simon Preložnik||Maja Remškar||Preprost dostavni sistem za omega-3 maščobne kisline||03.05.||05.05.||Jasmina Sedmak||Estera Merljak
|-
| Aneja Tuljak||Tina Gregorič||Stekleničke z biosenzorjem za detekcijo ''E.coli''||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.||Vida Špindler||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
|-
| Kaja Javoršek||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.

SB students resources

2015-02-08T23:01:09Z

Katarina Uršič: /* List of articles for presentation */

===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]]. Becskei ''et al''., EMBO J, 2001 - Andreja Bratovš
#[[Chemical synthesis of poliovirus cDNA: Generation of infectious virus in the absence of natural template]]. Jeronimo Cello ''et al''., Science,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. You et al, Nature (2004)[http://wiki.fkkt.uni-lj.si/index.php/7.Programmed_population_control_by_cell-cell_communication_and_regulated_killing] - Alja Zottel
#[[Gene regulation at the single-cell level]] Rosenfeld et al., Science, 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]]. Balagadde ''et al.'', ''Science'', 2005 - Jana Verbančič
#[[Tuning genetic control through promoter engineering]], Hal Alper ''et al''., PNAS, 2005 - Špela Pohleven
#[[Production of the antimalarial drug precursor artemisinic acid in engineered yeast ]]. Ro ''et al''., ''Nature''., 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]]. Dueber ''et al''., Nature Biotechnology, 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]], Miller ''et al'', ''Nature Biotechnol''., 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

Gene regulation at the single-cell level

2015-02-08T22:57:10Z

Katarina Uršič: New page: Rosenfeld, N., Young, J. W., Alon, U., Swain, P. S., & Elowitz, M. B. (2005). [http://www.sciencemag.org/content/307/5717/1962.full Gene regulation at the single-cell level.] Science 307 (...

Rosenfeld, N., Young, J. W., Alon, U., Swain, P. S., & Elowitz, M. B. (2005). [http://www.sciencemag.org/content/307/5717/1962.full Gene regulation at the single-cell level.] Science 307 (2005), 1962–1965].

In this paper I will present you »Gene regulation at the single-cell level« written by Nitzan Rosenfeld, Jonathan W. Young, Uri Alon, Peter S. Swain and Michael B. Elowitz. Article was published in Science in year 2005. Currently it can only be accessed after free registration on the Science web page.

REGULATION OF GENE EXPRESSION
----

Regulation of gene expression is necessary for maintaining cell autonomy. Organism from bacteria to mammals use it for adjusting to different internal and external environmental conditions while multicellular organism also use gene regulation for developing different cell types and tissue formation. Regulation of gene expression is a demanding task, the right genes have to be expressed at the right time, rate and at the right place.
In prokaryotes gene expression is usually managed by regulation of transcription. Cells can regulate the activity of already made proteins or they can alter the production rate by switching gene on or off. Operators act like a switch, they can enable the binding of RNA polymerase to promoter or not and therefore determine the fate of a gene transcription. Operons are under the influence of proteins made by regulatory genes. Repressor proteins can switch the operon off by binding to operator and preventing the beginning of transcription. There is a high level of specificity between regulatory proteins and operators, not all regulatory proteins can bind to all operator binding spots. Usually regulatory proteins are under control of constitutive promoters and are always expressed at least in small amounts. Binding or regulatory proteins is reversible, bigger expression rate of active regulatory proteins result in longer duration of operator state whether it be on or off state. Regulatory proteins are also often allosteric, present in active or inactive shape. Some need additional small molecules (corepressors or coactivators), that bind on them and then together can make a switch in expression. Roughly we can divide gene regulation into positive and negative. In positive gene regulation regulatory protein acts like an activator and its direct binding to DNA triggers transcription of a gene by increasing the affinity that RNA polymerase has for promoter. An example would be catabolite activator protein and its effect on lac operon. In negative gene regulation regulatory gene acts as a repressor and by binding to operator it turns the transcription of a gene off. Negative gene regulation can be preformed via repressible or inducible operons. Example of a repressible operon is trp operon which is usually active but it can be repressed when trp repressor and corepressor tryptophan bind together on its operator. Example of inducible operon is again lac operon. Repressor protein lacI binds to operator in the absence of lactose and prevents transcription of lac operon genes. When allolactose is present it acts as an inducer that inactivates the repressor protein (Campbell & Reece, 2008).
Eukaryotic gene expression regulation also strongly relies on regulation of transcription, based on the function of transcription factors similar to that in prokaryotes. In addition gene expression can also be regulated by changes in chromatin structure organization via histone modification and DNA methylation. Tails of histones can be acylated which loosens chromatin structure and enables transcription since transcriptional factors have easier access to DNA. Acetylaton neutralizes the positive charge of lysins and prevents binding of nucleosomes together. Other chemical groups can be attached to histone tails as well for example attachment of methyl group can have the opposite effect as it can promote chromatin to condensate. Methylation of cytosine can also help inactivate DNA and therefore lowers the expression rate of certain genes. Experiments also showed the effect of condensed heterochromatin on repression of gene expression. Transcription however is not the only factor determining the rate of gene production where we measure the actual amount of functional protein. When dealing with eukaryotes we have to take into consideration RNA processing such as alternative RNA splicing, speed of mRNA degradation in cytoplasm, initiation of translation which can be blocked by regulatory proteins that prevent binding of mRNA and ribosome and lastly the processing and degradation of protein (Campbell & Reece, 2008).

Not only is gene regulation important for adequate cell functioning, it is also a major factor in maintaining the proper functioning of synthetic genetic circuits. There is still a great lack of knowledge for building and maintaining complex, long term sustainable genetic networks that could have a potential use in real life applications. It is important to understand and characterize new parts and modules, model the behavior of synthetic networks and study the in vivo interactions between parts in a synthetic network. Many biological parts and interactions between them are still not well understood so synthetic networks often base on similar parts. New parts that show great promise are engineered zinc fingers and nucleic-acid-based parts. At single molecule level advanced technology enables direct ways to observe the state of each molecule. In vivo measurements cannot be performed that way because of the lack of technology. Currently the most promising development is in the field of microfluidic devices but the problem is the absence of reliable sensors (Lu, Khali3, & Collins, 2010).

INTRODUCTION TO EXPERIMENT
----

Rosenfeld et al. generated »lambda cascade« strains of E. coli. Bacteriophage lambda is a temperate phage meaning it can reproduce inside the bacteria via lysogenic or lytic cycle. They decided to characterized its PR promoter. In natural conditions, cI repressor protein maintains lysogenic cycle. It represses transcription from PR promoter by binding to one of the three binding sites OR1, OR2 or OR3 and preventing the expression of lytic genes. cI has the highest affinity for OR1 site but after cI dimerisation the affinity for OR2 increases too. Affinity for site OR3 is low and cI only binds when present in high concentrations (Bakk, Metzler, & Sneppen, 2004).
At the time that Rosenfeld et al. decided to performed the experiments models predicted gene regulation function (GRF) with the help of in vitro data. GRF is usually presented in a form of a graph with active transcription factor on the x axis and production rate on y axis. Due to many disadvantages authors decided to measure GRF in vivo, using lambda cascade strains of E. coli and a point mutation OR*-lambda cascade strain of E. coli. This designed lambda strain contained a fusion protein made from yellow fluorescent protein (YFP) and a repressor protein cI under control of a constitutive Ptet tetraycylin promoter. Ptet promoter can be repressed by TetR under PC promoter control and induced by tetracyclin or anhydrocyclin (aTc) [http://www.sciencemag.org/content/307/5717/1962/F1.expansion.html (Fig. 1B)]. cI acts as a repressor of PR promoter, that controls transcription of a cyan fluorescent protein (CFP). By constructing this genetic circuit they were able to simultaneously monitor and measure the amount of repressor protein and its target gene protein in time simply by measuring fluorescence (Rosenfeld et al., 2005).

REGULATION DILUTION EXPERIMENT
----

GRF tells us how production rates of a protein vary at different repressor concentrations therefore authors of the original paper had to find a way to systematically change it's concentration. To answer that problem they used a regulator dilution method. During the cell growth repressor production shuts off and as the volume of that cell increases, concentration of repressor decreases. Repressor also gets diluted by cell division. [http://www.sciencemag.org/content/307/5717/1962/F1.expansion.html Fig. 1C)]shows a graphic representation of regulator dilution experiment, showing us the fluctuation in concentration of cI-YFP and CFP over time (stated in cell cycles). Under normal conditions, TetR blocks cI-YFP production and cell express CFP under control of PR promoter. By adding aTc cells start to produce cI-YFP, which represses production of CFP as shown in [http://www.sciencemag.org/content/307/5717/1962/F1.expansion.html Fig. 1C]. Once aTc is no longer added, TetR represses production of cI-YFP so its concentration decreases and we reach additional dilution with each cell cycle as the cells grow and divide. Less of cI-YFP means less repression of CFP production and once cI-YFP levels decrease to a certain value, it can no longer efficiently repress cfp gene, CFP concentration indeed rises with each cell cycle. [http://www.sciencemag.org/content/307/5717/1962/F1.expansion.html Fig. 1D] shows regulator dilution experiment with OR2*-lambda cascade strain, that contains an OR2 point mutation which was made with the help of site-directed mutagenesis of the PR promoter [http://www.sciencemag.org/content/307/5717/1962/rel-suppl/cf6028dbca8cd6b5/suppl/DC1 (Fig. S3)]. Red represents cl-YFP and green represents CFP. Each snapshot shows time in minutes. With the help of fluorescent time-lapse microscopy ([http://www.sciencemag.org/content/307/5717/1962/F1.expansion.html Fig 1D], [http://www.sciencemag.org/content/307/5717/1962/rel-suppl/cf6028dbca8cd6b5/suppl/DC1 Fig. S1, Movie S1 and S2]) and computational image analysis they managed to form family trees for cells in microcolonies [http://www.sciencemag.org/content/307/5717/1962/rel-suppl/cf6028dbca8cd6b5/suppl/DC1 (Fig. S2)]. They then quantify the level of cI-YFP and CFP over time for each cell lineage and then calculated at what rate CFP was being produced (shown in [http://www.sciencemag.org/content/307/5717/1962/F2.expansion.html Fig 2A]) with level of cI-YFP on x axis and rate of CFP production on y axis. With the use of fluorescence information and cell family trees they managed to calibrate biochemical parameters with a new technique using binomial errors in protein partitioning. They compared levels of fluorescence of a sister cell pair just after the division. Since there is an equal possibility in cI-YFP molecule going in each sister cell, distribution of CI-YFP fluorescence was binomial. With the help of a single parameter fit they managed to estimate how much of detected fluorescence belonged to a single CI-YFP molecule. By calculating partitioning errors in CI-YFP distribution calibration was possible despite cellular autofluorescence. Binomial errors in partitioning helped find the apparent fluorescent intensity of one independently segregating fluorescent particle vy [http://www.sciencemag.org/content/307/5717/1962/F2.expansion.html(Fig. 2B)]. If a cell contained Ntot fluorescent particles, total fluorescence is Ytot=vy*Ntot. When a cell divides, sister cells gets N1 and N2 particles (Ntot=N1+N2). Based on the assumption that the segregation of particles is independent the segregation becomes binomial and satisfies the formula, used in mathematical model. [http://www.sciencemag.org/content/307/5717/1962/F3.expansion.html Fig. 3] shows results (3A shows mean GRF and 3B shows individual data) for PR and PR (OR2*) point mutation strain. Unknown regulation functions are often represented by Hill functions f(R) = β/[1 + (R/kd)n]. β is the maximal production rate, kd is a concentration of repressor that yields half-maximal expression and n is the degree of effective cooperativity in repression. When compared to data, measured in vivo, function fitted well ([http://www.sciencemag.org/content/307/5717/1962/F3.expansion.html Fig 3], [http://www.sciencemag.org/content/307/5717/1962.full#T1 table 1]). In vivo kd value was comparable to kd value estimated in previous studies. The effective cooperativity value (n>1) could be explained with cI repressor molecules dimerisation and interactions of repressor at neighboring sites. They also compared biomechanical parameters in PR and PR(OR2*) strain and found some differences. In PR strain, n is significantly increased and kd is significantly reduced ([http://www.sciencemag.org/content/307/5717/1962.full#T1 table 1]). PR (OR2*) strain contained a mutation of a binding site, which could prevent cI from binding or lowered its affinity for cI and therefore we would need higher repressor concentration to achieve the same results as in PR strain. After observing the mean regulation function of wild type PR and its mutated variant OR2* they wanted to further investigate deviations from the mean GRF. Standard deviations of CFP production rates was about 55% of the mean GRF for any given cI-YFP concentration (Rosenfeld et al., 2005).

STANDARD DEVIATION TESTS AND CELL NOISE
----

There are many possible reasons for variations like this, from differences in environment of the cells, changes in gene copy number or noises.
Noise causes differences even in identical cells or organisms. With molecules that are present within a cell in a large number, reactions can be predicted. That is however not possible with molecules like DNA or some proteins that are presented in a fewer copies per cell and can be influenced by random stochastic fluctuations. Noise could be a reason for reduced preciseness of cellular processes by effecting gene expression. Variation of gene expression can have its source in four main factors: differences in environment, mutations, differences in inner cell environment and stochasticity related to molecules with small copy number per cell. Noise is usually measured with the help of fluorescent proteins and is defined as the ratio between standard deviation and the mean value. Amplitude of noise is controlled by genetic factors, rate of transcription and regulatory dynamics (Raser and O'Shea, 2005).
Intrinsic noise comes from biochemical reactions at genes, consequently changing the expression rates of two identical copies of gene. Extrinsic noise comes from differences in rates of cellular components. The difference in expression proteins can be contributed to intrinsic noise such as protein degradation, fluctuations in the amount of mRNA or different levels of promoter-biding. Extrinsic noise has the same effect on expression of both proteins inside a cell but effects different cells differently (differences in environment, concentration and activity of trancriptional factors etc.) extrinsic noise can be further divided into global and specific noise. Global noise comes from fluctuations in basic reactions that effect the whole cell. Specific noise originates in fluctuations of a factor that only effects a specific gene or pathway (Raser and O'Shea, 2005).
Measuring of noise and determining whether its intrinsic or extrinsic could be done by comparing expression of two fluorescent proteins under identical regulation in the same cell. This two-reporter method was developed by Michael B. Elowitz. With coworkers they observed protein expression under control of identical promoters on the same E. coli chromosome which enabled them to distinguish between the two main types of noise. Intrinsic noise was measured by comparing the amount of both fluorescent proteins, CFP and YFP, produced in a single cell. Effect of extrinsic noise was observed when production rates of both proteins were the same for a single cell but varied among multiple cells (Elowitz et al., 2002).
Studies confirm that the dominant effect of global extrinsic noise over intrinsic noise and is a major source of variability between cells. Overall effect of noise on cells depends on the degree and the recurrence of noise. Time is an important factor, the longer the endurance, the greater the effect can be. Evolutionary speaking, noise can have a positive or a negative impact, intrinsic noise can increase the expression of different allele combinations and therefore increases phenotypic variability. In a stressful situations or change in environment, some cells could potentially adapt better than other or there could be opposite response (Raser and O'Shea, 2005).
Noise can effect an entire genetic network and can be amplified. A study, made by Hooshangi et al. showed how effect of noise increases with the complexity of genetic circuit. They designed three circuits, each with one additional transcriptional step in a regulatory cascade. What they concluded is that the longer the cascade, the bigger the cell variability and coordination of cell responses reduced (Hooshangi, Thiberge, & Weiss, 2005).
Rosenfeld et al. tested the environment possibility by comparing different microcolonies that were induced at different cell densities but no significant changes in GRF were detected ([http://www.sciencemag.org/content/307/5717/1962/rel-suppl/cf6028dbca8cd6b5/suppl/DC1 Fig. S6]). Next they analyzed how the increased gene copy number during DNA replication/cell division would affect GRF. The results showed strong correlation, newly divided cells produced less CFP than cells just prior to division. Even with that taken into consideration, standard deviation was still around 40 % of mean GRF after normalizing production rates of CFP to an average phase of a cell cycle. Lastly they tested effects of intrinsic and extrinsic noise.

SYMMETRIC BRANCH STRAIN TEST
----

To find out the origin of fluctuations, they designed a »symmetric branch« strain (Fig. 4D, movie S3), similar to one mentioned before, to help them distinguish between intrinsic and extrinsic noise. Symmetric branch strain of E. coli produced YFP and CFP under control of two identical PR promoters, both regulated by cl-YFPY66F repressor protein ([http://www.sciencemag.org/content/307/5717/1962/F4.expansion.html Fig. 4D]). cI-YFPY66F gene contained a mutation made with the help of site-directed mutagenesis. They introduced a single point mutation which changed the tyrosine at YFP position 66 to phenylalanine which prevented repressor fluorescence so the only YFP fluorescence detected came from the YFP under PR control and not the fusion repressor protein. After measuring the expression of both fluorescent protein genes by measuring fluorescence for each cell, results concluded that the difference in production rates is bigger because of extrinsic noise (intrinsic noise played a role too). Extrinsic noise has bigger effect on the difference in production rates than intrinsic noise (about 35 % versus 20 %). Because they quantified the extrinsic noise at known repressor concentration, the only fluctuations measured are those of global cellular components. Analysis of extrinsic noise is also more precise because of its dynamic observation – »measuring in the rate of protein expression« (Rosenfeld et al., 2005).
Different paced fluctuations affect genetic networks in different ways. One way to characterize fluctuations in by τcorr, their autocorrelation time. If a fluctuation is longer than the length of a cell cycle, it can accumulate and produce significant effects whereas short term fluctuations (much shorter than a cell cycle) have smaller effects as they even out. Authors divided their measurements into three categories ([http://www.sciencemag.org/content/307/5717/1962/F4.expansion.html Fig. 4A-C]): fast fluctuations similar to intrinsic noise; periodic DNA replication related fluctuations and aperiodic fluctuations, similar to extrinsic noise. When comparing mean GRF with a selected single cell lineage lambda, we can see irregularities ([http://www.sciencemag.org/content/307/5717/1962/F3.expansion.html Fig. 3B]) with autocorrelation time around 40 minutes ([http://www.sciencemag.org/content/307/5717/1962/F4.expansion.html Fig. 4E]) which is close to the length of a cell cycle (around 45±10 minutes). Therefore most fluctuations persist for about one cell cycle (whether it be overexpression or underexpression of CFP). These observed fluctuations are too slow to be classified as a consequence of intrinsic noise (which has autocorrelation time under 10 minutes). As we're modeling genetic circuits whether they be natural or synthetic, we must take extrinsic fluctuations into consideration (Rosenfeld et al., 2005).

CONCLUSION
----

Authors of original article successfully characterize lambda promoter, in vivo calibrated biochemical parameters using novel techniques and characterized the effect of extrinsic and intrinsic noise. GRF efficiency of a cell is determined by various factors and when constructing a genetic network it is crucial to take that into consideration.

References:
----

Campbell, N. A., & Reece, J. B. (2008). Biology 8th edition. San Francisco: Pearson Benjamin Cummings. 351-364.

Bakk, A., Metzler, R., & Sneppen, K. (2004). Sensitivity of OR in phage lambda. Biophysical Journal, 86(January), 58–66. doi:10.1016/S0006-3495(04)74083-7

Elowitz, M. B., Levine,A. J., Siggia,E. J., & Swain P. S. (2002). Stochastic gene expression in a single cell. Science (New York, N.Y.), 297(5584): 1183-1186. doi:10.1126/science.1070919

Hooshangi, S., Thiberge, S., & Weiss, R. (2005). Ultrasensitivity and noise propagation in a synthetic transcriptional cascade. Proceedings of the National Academy of Sciences of the United States of America, 102(10), 3581–6. doi:10.1073/pnas.0408507102

Lu, T. K., Khali3, A. S., & Collins, J. J. (2010). Next-Generation Synthetic Gene Networks. Nat Biotechnol, 27(12), 1139–1150. doi:10.1038/nbt.1591.Next-Generation

Raser, J. M., & O'Shea, E. K. (2005). Noise in gene expression: origins, consequences and control. Science (New York, N.Y.), 309(5743): 2010–2013. doi:10.1126/science.1105891

Rosenfeld, N., Young, J. W., Alon, U., Swain, P. S., & Elowitz, M. B. (2005). Gene regulation at the single-cell level. Science (New York, N.Y.), 307(2005), 1962–1965. doi:10.1126/science.1106914

Combined Cancer Gene Therapy

2014-01-10T23:36:54Z

Katarina Uršič:

'''In Vitro and In Vivo Effect of 5-FC Combined Gene Therapy with TNF-α and CD Suicide Gene on Human Laryngeal Carcinoma Cell Line Hep-2'''

1 UVOD

Rak na grlu je ena najpogostejših oblik raka na območju glave in vratu. Manj kot 60% pacientov preživi več kot 5 let po terapiji. V študiji so preiskovali učinek kombinirane genske terapije z eksogenim tumor nekrotskim faktorjem α (TNF-α) in citozin deaminazo (CD) samomorilskim genom na celično linijo Hep-2 celic raka na grlu.
TNF-α je citokin, ki lahko inhibira napredovanje rasti tumorja po različnih signalnih poteh.
CD/5-fluorocitozin sistem: CD pretvori 5-FC v toksičen 5-FU, ki povzroča motnje v biosintezi DNA.

2 MATERIALI, METODE IN REZULTATI

• Kloniranje tarčnih genov in konstrukcija vektorjev:
Izvedli so colony PCR z E.coli JM109 in izolirali DNA. Iz človeških levkocitov so izolirali RNA in z RT-PCR pomnožili TNF-α gen. S PCR produkti CD gena in RT-PCR produkti TNF-α gena so izvedli gelsko elektroforezo.

V pcDNA 3.1(+) vstavili TNF-α in CD gen ter tako dobili pcDNA 3.1(+)-TNF-α in pcDNA 3.1(+)-CD.
Not I in Xho I sta rezala plazmid pIRES, IRES segment je bil izoliran z gelsko ekstrakcijo. pcDNA 3.1(+)-CD so razrezali, nato pa vstavili TNF-α segment in dobili pcDNA 3.1(+)-TNF-α-CD. Plazmid so ponovno razrezali, vstavili IRES in dobili še pcDNA3.1(+)-TNF-α-IRES-CD. Vektorje pcDNA 3.1(+)-CD, pcDNA 3.1(+)-TNF-α-CD in pcDNA3.1(+)-TNF-α-IRES-CD so nato rezali in produkte potrdili z gelsko elektroforezo.

• Transfekcija:
Opravili so liposomsko transfekcijo po protokolu za Lipofectamin 2000 . Hep-2 celice so transfecirali s pcDNA 3.1(+)-TNF-α-CD, pcDNA 3.1(+)-CD, pcDNA 3.1(+)-TNF-α in pcDNA 3.1(+). Po transfekciji so Hep-2 inkubirali 2 tedna ob dodatku G418. Pozitivne klone so namnožili in jih poimenovali Hep-2/TIC, Hep-2/CD, Hep-2/TNF-a in Hep-2/0.

• Potrditev genske ekspresije s RT-PCR:
Iz Hep-2/TIC, Hep-2/CD, Hep-2/TNF-α in Hep-2/0 so izolirali RNA. Izvedli so RT-PCR in produkte uporabili za gelsko elektroforezo.

• In vitro citocidni učinek:
Hep-2/TIC, Hep-2/CD, Hep-2/TNF-α, Hep-2/0 in starševske Hep-2 celice so inokulirali v ploščo z 96 vdolbinicami (18 za vsak tip celic). Po inkubaciji so različne koncentracije 5-FC (3 vdolbinice za vsako koncentracijo). Po 96ih urah so dodali MTT in inkubirali še 4 ure. Odstranili so supernatant, dodali DMSO in z metodo ELISA izmerili absorbanco.
5-FC je imel največji učinek na Hep-2/TIC.

• "Bystander" učinek:
Mešanice z različnimi vsebnostmi celičnih linij so dodali 1 mM 5-FC in po inkubaciji določili stopnjo preživetja z metodo ELISA.
"Bystander" učinek so opazili v vseh petih tipih celic, najbolj v Hep-2/TIC.

• Analiza apoptoze:
Hep-2/TIC, Hep-2/CD, Hep-2/TNF-α in Hep-2/0 so dodal 1 mM 5-FC in s pretočno citometrijo izmerili stopnjo apoptoze.
Stopnja apoptoze se je zvišala, stopnja proliferacije pa znižala; najbolj očitno pri Hep-2/TIC.

• Detekcija TNF-α proteina:
Hep-2/TIC, Hep-2/CD, Hep-2/TNF-α, Hep-2/0 so inokulirali v ploščo z 96 vdolbinicami. Po inkubaciji so zbrali supernatant in z ELISO izmerili absorbanco.
Najvišjo koncentracijo so zaznali pri Hep-2/TIC in Hep-2/TNF-α.

• Subkutani tumorski model:
Suspenzijo tumorskih celic so inokulirali v 53 miši, ki so jih naključno razporedili v štiri skupine: Hep-2/TIC (13 osebkov), Hep-2/CD (13 osebkov), Hep-2/TNF-α (13 osebkov) in Hep-2/0 (14 osebkov).
Osmi dan po inokulaciji so pričeli z zdravljenjem s 5-FC. Merili so velikost tumorjev ter izračunali volumen in čas podvojitve. Po 30ih dneh so miši usmrtili in izolirali tumorsko tkivo. Rezultati so pokazali, da produkti izražanja CD in TNF-α genov inhibirajo rast presajenih celic pri mišjem modelu.

Primerjali so tudi patološke spremembe v izoliranem tkivu tumorjev vseh štirih skupin in ugotovili razlike med skupinami.

3 ZAKLJUČEK

Študija je z in vivo in in vitro eksperimenti potrdila sinergistično delovanje kombinacije CD in TNF-α gena. Glede na rezultate sklepajo, da je kombinacija uspela delovati na tumorje preko več različnih poti in ima aditivne učinke; TNF-α inducira visoko prepustnost tumorske vaskulature in tako pomaga zbrati in skoncentrirati 5-FU v tumorsko tkivo.
Glede na rezultate so predpostavili, da bi 5-FC kombinirana genska terapija s TNF-α in CD samomorilnim genom lahko predstavljala učinkovito zdravljenje raka na grlu pri ljudeh v prihodnosti.

4 VIRI

Chai L-P, Wang Z-F, Liang W-Y, Chen L, Chen D, et al. (2013) In Vitro and In Vivo Effect of 5-FC Combined Gene Therapy with TNF-a and CD Suicide Gene on Human Laryngeal Carcinoma Cell Line Hep-2. PLoS ONE 8(4): e61136. doi:10.1371/journal.pone.0061136
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0061136

Combined Cancer Gene Therapy

2014-01-10T23:05:21Z

Katarina Uršič: In Vitro and In Vivo Effect of 5-FC Combined Gene Therapy with TNF-a and CD Suicide Gene on Human Laryngeal Carcinoma Cell Line Hep-2

'''In Vitro and In Vivo Effect of 5-FC Combined Gene Therapy with TNF-a and CD Suicide Gene on Human Laryngeal Carcinoma Cell Line Hep-2'''

1 UVOD

Rak na grlu je ena najpogostejših oblik raka na območju glave in vratu. Manj kot 60% pacientov preživi več kot 5 let po terapiji. V študiji so preiskovali učinek kombinirane genske terapije z eksogenim tumor nekrotskim faktorjem α (TNF-α) in citozin deaminazo (CD) samomorilskim genom na celično linijo Hep-2 celic raka na grlu.
TNF-α je citokin, ki lahko inhibira napredovanje rasti tumorja po različnih signalnih poteh.
CD/5-fluorocitozin sistem: CD pretvori 5-FC v toksičen 5-FU, ki povzroča motnje v biosintezi DNA.

2 MATERIALI, METODE IN REZULTATI

• Kloniranje tarčnih genov in konstrukcija vektorjev:
Izvedli so colony PCR z E.coli JM109 in izolirali DNA. Iz človeških levkocitov so izolirali RNA in z RT-PCR pomnožili TNF-α gen. S PCR produkti CD gena in RT-PCR produkti TNF-α gena so izvedli gelsko elektroforezo.

V pcDNA 3.1(+) vstavili TNF-α in CD gen ter tako dobili pcDNA 3.1(+)-TNF-α in pcDNA 3.1(+)-CD.
Not I in Xho I sta rezala plazmid pIRES, IRES segment je bil izoliran z gelsko ekstrakcijo. pcDNA 3.1(+)-CD so razrezali, nato pa vstavili TNF-α segment in dobili pcDNA 3.1(+)-TNF-α-CD. Plazmid so ponovno razrezali, vstavili IRES in dobili še pcDNA3.1(+)-TNF-α-IRES-CD. Vektorje pcDNA 3.1(+)-CD, pcDNA 3.1(+)-TNF-α-CD in pcDNA3.1(+)-TNF-α-IRES-CD so nato rezali in produkte potrdili z gelsko elektroforezo.

• Transfekcija:
Opravili so liposomsko transfekcijo po protokolu za Lipofectamin 2000 . Hep-2 celice so transfecirali s pcDNA 3.1(+)-TNF-α-CD, pcDNA 3.1(+)-CD, pcDNA 3.1(+)-TNF-α in pcDNA 3.1(+). Po transfekciji so Hep-2 inkubirali 2 tedna ob dodatku G418. Pozitivne klone so namnožili in jih poimenovali Hep-2/TIC, Hep-2/CD, Hep-2/TNF-a in Hep-2/0.

• Potrditev genske ekspresije s RT-PCR:
Iz Hep-2/TIC, Hep-2/CD, Hep-2/TNF-a in Hep-2/0 so izolirali RNA. Izvedli so RT-PCR in produkte uporabili za gelsko elektroforezo.

• In vitro citocidni učinek:
Hep-2/TIC, Hep-2/CD, Hep-2/TNF-α, Hep-2/0 in starševske Hep-2 celic so inokulirali v ploščo z 96 vdolbinicami (18 za vsak tip celic). Po inkubaciji so različne koncentracije 5-FC (3 vdolbinice za vsako koncentracijo). Po 96ih urah so dodali MTT in inkubirali še 4 ure. Odstranili so supernatant, dodali DMSO in z metodo ELISA izmerili absorbanco.
5-FC je imel največji učinek na Hep-2/TIC.

• "Bystander" učinek:
Mešanice z različnimi vsebnostmi celičnih linij so dodali 1 mM 5-FC in po inkubaciji določili stopnjo preživetja z metodo ELISA.
"Bystander" učinek so opazili v vseh petih tipih celic, najbolj v Hep-2/TIC.

• Analiza apoptoze:
Hep-2/TIC, Hep-2/CD, Hep-2/TNF-α in Hep-2/0 so dodal 1 mM 5-FC in s pretočno citometrijo izmerili stopnjo apoptoze.
Stopnja apoptoze se je zvišala, stopnja proliferacije pa znižala; najbolj očitno pri Hep-2/TIC.

• Detekcija TNF-α proteina:
Hep-2/TIC, Hep-2/CD, Hep-2/TNF-α, Hep-2/0 so inokulirali v ploščo z 96 vdolbinicami. Po inkubaciji so zbrali supernatant in z ELISO izmerili absorbanco.
Najvišjo koncentracijo so zaznali pri Hep-2/TIC in Hep-2/TNF-α.

• Subkutani tumorski model:
Suspenzijo tumorskih celic so inokulirali v 53 miši, ki so jih naključno razporedili v štiri skupine: Hep-2/TIC (13 osebkov), Hep-2/CD (13 osebkov), Hep-2/TNF-α (13 osebkov) in Hep-2/0 (14 osebkov).
Osmi dan po inokulaciji so pričeli z zdravljenjem s 5-FC. Merili so velikost tumorjev ter izračunali volumen in čas podvojitve. Po 30ih dneh so miši usmrtili in izolirali tumorsko tkivo. Rezultati so pokazali, da produkti izražanja CD in TNF-α genov inhibirajo rast presajenih celic pri mišjem modelu.

Primerjali so tudi patološke spremembe v izoliranem tkivu tumorjev vseh štirih skupin in ugotovili razlike med skupinami.

3 ZAKLJUČEK

Študija je z in vivo in in vitro eksperimenti potrdila sinergistično delovanje kombinacije CD in TNF-α gena. Glede na rezultate sklepajo, da je kombinacija uspela delovati na tumorje preko več različnih poti in ima aditivne učinke; TNF-α inducira visoko prepustnost tumorske vaskulature in tako pomaga zbrati in skoncentrirati 5-FU v tumorsko tkivo.
Glede na rezultate so predpostavili, da bi 5-FC kombinirana genska terapija s TNF-α in CD samomorilnim genom lahko predstavljala učinkovito zdravljenje raka na grlu pri ljudeh v prihodnosti.

4 VIRI

Chai L-P, Wang Z-F, Liang W-Y, Chen L, Chen D, et al. (2013) In Vitro and In Vivo Effect of 5-FC Combined Gene Therapy with TNF-a and CD Suicide Gene on Human Laryngeal Carcinoma Cell Line Hep-2. PLoS ONE 8(4): e61136. doi:10.1371/journal.pone.0061136
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0061136