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A novel adenoviral vector carrying an all-in-one Tet-On system with an autoregulatory loop for tight, inducible transgene expresion

2015-03-25T12:24:18Z

Edvinas Grauželis: New page: == Introduction == In this research article scientist introduce new Adenoviral vector with Tet-On loop regulation system. Research was made according the adenoviral “All-in-one” Tet-O...

== Introduction ==

In this research article scientist introduce new Adenoviral vector with Tet-On loop regulation system. Research was made according the adenoviral “All-in-one” Tet-On system issues to regulate gene expression. Although adenoviral vector is one of the most usable vectors in gene therapy, it was hardly compatible to the Tet-On system. There were used new promoters genes and proteins for better reconciliation. Primal idea for this research was suggested as a better way to coordinate gene expression since it would be more convenient.

== Methods and technologies ==

There were created four different new adenoviral Tet-On vectors with different promoters, length of the gene, silencers. Three from four construct have cytomegalavyrus promoter sequence, two of four have double frame reading points in the other words two promoters, all target genes will be poliadenylated in 3’ end. One of four has KRAB expression cassette comparible with Tet genes. All of these vectors were made by using PCR and restriction methods. E1 region was left for target genes.

There were used HEK 293 a human embryonic kidney cells, SW480 a human colon carcinoma cells and U87 MG a human glioblastoma cell line. Moreover they used CHO-K1 a Chinese hamster ovary cell line.

Dual system of Luciferase was used to normalize the cell number and transfection efficiency. After 48 hours of transfection, cells were collected for the test by using dual-luciferase kits provided by Promega. The normalized Luciferase activity was obtained by using the formula: Normalized Luciferase value equal Fly luciferase/ Renilla ludiferase value.

Adenovirus was produced in the HEK293 cell line. Adenovirus “all-in-on” plasmid was made by transfection as a back bone using adenovirus plasmid and linearized constructed sequence. After ten days viral lysates were harvest and propagated in HEK293 and were purified using cesium chloride. The virus particle titers were detected by spectrophotometry at an absorbance at 260nm.

For counting the statistics were used SPSS program packet. Groups were analyzed by one way analysis of variance between groups (ANOVA/LSD). With p value less than 0,05 for significant difference.

== Results ==

The major problem with an adenoviral vector carrying a Tet-On system is the suboptimal regulation of transgene expression. For this reason two newly constructed vectors have a new version of code of the Dox sensitive transactivator.

A novel system of “All-in-one” was evaluated by eGFP in comparing with control. Control in the absence of Dox gene expression was suppressed as well as in the presence of 2µl/ml of Dox. The two most important criteria for evaluating a regulation system are the regulation factor and the maximum induced transgene expression level. To measure these two criteria luciferase gene was cloned into each plasmid. During the experiments it was seen that in the absence of Dox new system is suppressing the genes in the presence of the Dox visible reaction was seen. Measuring the luciferase activity showed significant difference between two phases. Moreover, maximum gene expression level was reach after forty hours of induction of 2µl/ml Dox.

To investigate whether the novel Tet-On system could be able to regulate medical related genes approaches. Into the E1 region of plasmid was inserted TRAIL gene. TRAIL is anticancer agent that selectively induces apoptosis in variety of cancer cells although the TRAIL is toxic for normal cells. For this reason there is some need to strictly regulate the gene expression of the TRAIL in the targeted cell.

Two novel “all-in-one” systems were presented to the host cells, one of the virus show low level of infecting and low cell death ratio due to TRAIL system inside. Another virus has greater infecting ratio and still low level of cell death, both virus results was in the absence of the Dox. In presence of a Dox cell death ratio increased. Although one of the virus loop have revealed greater suppressor activity in the presence of the Dox.

== Conclusion ==

In this study was successfully constructed functional tetracycline inductive adenoviral vector system. Further researches will allow searching for the best properties of “all-in-one” system for better results; moreover this is the first adenovirus-based, all-in-one Tet-on system with large packing size up to 2kb.

MBT seminarji 2015

2015-03-25T12:20:02Z

Edvinas Grauželis:

'''Seznam seminarjev iz Molekularne biotehnologije v študijskem letu 2014/15'''

Tabela za razpored po tednih bo objavljena v spletni učilnici, vanjo pa se vpišite tudi za kratke predstavitve novic (3 min, dvakrat v semestru). Na tej strani bo samo seznam odobrenih člankov za seminar in povezave do člankov in do povzetkov, ki jih morate objaviti najkasneje tri dni pred predstavitvijo (ponedeljek oz. torek). Angleški naslov prevedite tudi v slovenščino - to bo naslov povzetka, ki ga objavite na posebni strani, tako kot so to naredili kolegi pred vami (oz. lani).

Način vnosa:

# The importance of ''Arabidopsis'' glutathione peroxidase 8 for protecting ''Arabidopsis'' plant and ''E. coli'' cells against oxidative stress (A. Gaber; GM Crops & Food 5(1), 2014; http://dx.doi.org/10.4161/gmcr.26979) Pomen glutation peroksidaze 8 iz repnjakovca za zaščito rastline ''Arabidopsis thaliana'' in bakterije ''Escherichia coli'' pred oksidativnim stresom. Janez Novak, 15. marca 2014
(slovenski naslov povežite z novo stranjo, na kateri bo povzetek)

'''Naslovi odobrenih člankov po temah:'''

'''Gensko spremenjene rastline''' 

# Successful high-level accumulation of fish oil omega-3 long-chain polyunsaturated fatty acids in a transgenic oilseed crop (Ruiz-Lopez, N., et al; The plant journal 77, 198-208, 2014; http://www.ncbi.nlm.nih.gov/pubmed/24308505). [[Uspešna priprava gensko spremenjene oljne rastline z visoko vsebnostjo omega-3 polinenasičenih maščobnih kislin.]] Petra Malavašič, 20. marca 2015
#A simpliﬁed and accurate detection of the genetically modiﬁed wheat MON71800 with one calibrator plasmid (Jae Juan, S.,et al; Food Chemistry 176, 1-6, ;http://www.sciencedirect.com.nukweb.nuk.uni-lj.si/science/article/pii/S03088146140196572015 [[Poenostavljena in točna detekcija gensko spemenjene pšenice MON71800 z enim kalibratorskim plazmidom]] Matej Lesar, 20.marca 2015

'''Gensko spremenjene živali''' 

# [[A novel adenoviral vector carrying an all-in-one Tet-On system with an autoregulatory loop for tight, inducible transgene expresion]] (H. Chen; et all.; BMC Biotechnology 2015, 15:4, doi:10.1186/s12896-015-0121-4; http://www.biomedcentral.com/1472-6750/15/4). Edvinas Grauželis, 27. marca 2015 (in English)
# Production of functional active human growth factors in insects used as living biofactories (B. Dudognon, et al; Journal of Biotechnology 184, 229–239, 2014; http://dx.doi.org/10.1016/j.jbiotec.2014.05.030). [[Proizvodnja funkcionalno aktivnih človeških rastnih faktorjev v insektih uporabljenih kot žive biotovarne]] Maxi Sagmeister, 27. marca 2015

'''Okolje''' 
# Bioremediation of pesticide contaminated water using an organophosphate degrading enzyme immobilized on nonwoven polyester textiles (Yuan Gao ''et al.'', Enzyme and Microbial Technology, vol. 54, pages 38-44, 10.1.2014, http://www.sciencedirect.com/science/article/pii/S0141022913002044). Bioremediacija s pesticidi okužene vode z uporabo encima, ki razgrajuje organofosfate in je vezan na netkan poliesterski tekstil. Mitja Crček,
# Biodegradation of atrazine by three transgenic grasses and alfalfa expressing a modified bacterial atrazine chlorohydrolase gene (A. W. Vail ''et al.''; Transgenic Research, 29. 11. 2014; http://link.springer.com/article/10.1007/s11248-014-9851-7). Biorazgradnja atrazina s tremi transgenskimi travami in alfalfo, ki izražajo gen za modificirano bakterijsko atrazin klorohidrolazo. Mirjam Kmetič,

'''Terapevtiki''' 
# Mind-controlled transgene expression by a wireless-powered optogenetic designer cell implant (M. Folcher; Nature Communications 5, 1–11, 2014; http://www.nature.com/ncomms/2014/141111/ncomms6392/full/ncomms6392.html) Z EEG nadzorovano izražanje transgena preko brezžično napajanega optogenetskega celičnega vsadka. Luka Smole, 9. marca 2015

'''Encimi''' 

# Construction of efficient xylose utilizing ''Pichia pastoris'' for industrial enzyme production (Li ''et al''; Microbial Cell Factories 14:22, 1-10, 2015; http://www.microbialcellfactories.com/content/14/1/22). Priprava ''Pichie pastoris'', ki učinkovito uporablja ksilozo, za industrijsko proizvodnjo encimov. Špela Tomaž

'''Protitelesa''' 

# Functional mutations in and characterization of VHH against Helicobacter pylori urease (R. Hoseinpoor ''et al''; Applied Biochemistry and Biotechnology 172, 3079-3091, 2014; http://link.springer.com/article/10.1007/s12010-014-0750-4). Funkcionalne mutacije in karakterizacija VHH proti ureazi ''Helicobacter pylori''. Marko Radojković, 9. marca 2015
#Ethanol precipitation for purification of recombinant antibodies (A. Tscheliessnig ''et al''; Journal of Biotechnology 188, 17-28, 2014; http://www.sciencedirect.com/science/article/pii/S0168165614007810). Čiščenje rekombinantnih protiteles z obarjanjem z etanolom. Urška Rauter, 10. marca 2015

'''Cepiva''' 

# A novel “priming-boosting” strategy for immune interventions in cervical cancer (S. Liao et al.; Molecular Immunology 64, 295-305, 2015, http://www.sciencedirect.com/science/article/pii/S0161589014003460. Nova "priming-boosting" strategija za imunsko posredovanje pri raku materničnega vratu. Anita Kustec, 11. marca 2015
# Potentiation of anthrax vaccines using protective antigen-expressing viral replicon vectors (H.C. Wang et al.; Immunology letters 163, 206-213, 2015, http://www.ncbi.nlm.nih.gov/pubmed/25102364 ) Izboljšava cepiv proti antraksu z uporabo iz virusnih replikonov izvedenih vektorjev, ki omogočajo izražanje zaščitnega antigena. Daša Pavc

'''Male molekule in polimeri''' 
Iza Ogris 

# Chromosomal integration of hyaluronic acid synthesis (''has'') genes enhances the molecular weight of hyaluronan produced in ''Lactococcus lactis'' (R. V. Hmar et al; Biotechnol. J. 9 (12), 2014; http://dx.doi.org/10.1002/biot.201400215) Integracija genov za sintezo hialuronske kisline v kromosom bakterije ''Lactococcus lactis'' izboljša sintezo visokomolekularne hialuronske kisline. Maja Grdadolnik.

'''Pretvorba biomase''' 

'''Metabolično inženirstvo''' 

# Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived biofuels and chemicals (Weerawat Runguphana, Jay D. Keasling; Metabolic Engineering, vol 21, January 2014, Pages 103–113; http://www.sciencedirect.com/science/article/pii/S1096717613000670). Metabolično inženirstvo ''Saccharomyces cerevisiae'' za proizvodnjo derivatov maščobnih kislin, ki so primerni za biogorivo in kemikalije. Dominik Kert, 9. marca 2015

# Metabolic engineering of Klebsiella pneumoniae for the production of cis,cis-muconic acid (Jung,H.-M. Jung,M.-Y. Oh, M.-K.;Applied Microbiology and Biotechnology, Published online: 14 February 2015; http://link.springer.com/article/10.1007/s00253-015-6442-3). Metabolno inženirstvo Klebsiella pneumoniae za produkcijo cis,cis-mukonične kisline. Jure Zabret, 15. marca 2015
'''Biološki viri energije'''

User:Edvinas Grauželis

2015-03-25T12:10:40Z

Edvinas Grauželis: New page: A student of Vytautas Magnum University. Erasmus student in Ljubljana University.

A student of Vytautas Magnum University. Erasmus student in Ljubljana University.

MBT seminarji 2015

2015-03-09T12:43:18Z

Edvinas Grauželis: