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Optimization of a genome-wide disordered lentivector-based short hairpin RNA library

  • Genomics. Transcriptomics. Proteomics
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Abstract

To obtain a whole genome library that suppresses the total diversity of human mRNAs, lentiviral vector constructs and a short hairpin RNA (shRNA) expression cassette were optimized. The optimization of the vector increased the virus titer in preparations by 15–20 times. A simple shRNA structure with a 21-bp stem proved to be the most effective. Lentivector-based shRNA expression constructs were obtained by using puro R, copGFP, or H-2K k as a selectable marker. The efficiency of the optimized library was demonstrated when screening for shRNAs reactivating the tumor suppressor p53 in HeLa cells. Cells carried a reporter construct ensuring p53-responsive synthesis of a fluorescent protein, which allowed selection of cells with reactivated p53 by flow cytometry.

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References

  1. Dykxhoorn D.M., Novina C.D., Sharp P.A. 2003. Killing the messenger: Short RNAs that silence gene expression. Nature Rev. Mol. Cell Biol. 4, 457–467.

    Article  CAS  Google Scholar 

  2. Fire A., Xu S., Montgomery M.K., Kostas S.A., Driver S.E., Mello C.C. 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 391, 806–811.

    Article  PubMed  CAS  Google Scholar 

  3. Ngo H., Tschudi C., Gull K., Ullu E. 1998. Doublestranded RNA induces mRNA degradation in Trypanosoma brucei. Proc. Natl. Acad. Sci. USA. 95, 14687–14692.

    Article  PubMed  CAS  Google Scholar 

  4. Yang S., Tutton S., Pierce E., Yoon K. 2001. Specific double-stranded RNA interference in undifferentiated mouse embryonic stem cells. Mol. Cell. Biol. 21, 7807–7816.

    Article  PubMed  CAS  Google Scholar 

  5. Hutvágner G., McLachlan J., Pasquinelli A.E., Bálint É., Tuschl T., Zamore P.D. 2001. A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science. 293, 834–838.

    Article  PubMed  Google Scholar 

  6. Moss E.G. 2002. MicroRNAs: Hidden in the genome. Curr. Biol. 12, R138–R140.

    Article  PubMed  CAS  Google Scholar 

  7. Bartel D.P. 2004. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell. 116, 281–297.

    Article  PubMed  CAS  Google Scholar 

  8. Berstein E., Caudy A.A., Hammond S.M. Hannon G.J. 2001. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature. 409, 363–366.

    Article  CAS  Google Scholar 

  9. Caplen N.J., Parrish S., Imani F., Fire A., Morgan R.A. 2001. Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc. Natl. Acad. Sci. USA. 98, 9742–9747.

    Article  PubMed  CAS  Google Scholar 

  10. Lee Y., Ahn C., Choi H., Kim J., Yim J., Lee J., Provost P., Radmark O., Kim S., Kim V.N. 2003. The nuclear RNase III Drosha initiates microRNA processing. Nature. 425, 415–419.

    Article  PubMed  CAS  Google Scholar 

  11. Hammond S.M., Bernstein E., Beach D., Hannon G.J. 2000. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature. 404, 293–296.

    Article  PubMed  CAS  Google Scholar 

  12. Carmell M.A., Xuan Z., Zhang M.Q., Hannon G.J. 2002. The Argonaute family: Tentacles that reach into RNAi, developmental control, stem cell maintenance, and tumorigenesis. Genes Dev. 16, 2733–2742.

    Article  PubMed  CAS  Google Scholar 

  13. Sledz C.A., Williams B.R.G. 2005. RNA interference in biology and disease. Blood. 106, 787–794.

    Article  PubMed  CAS  Google Scholar 

  14. Hammond S.M. 2005. Dicing and slicing: The core machinery of the RNA interference pathway. FEBS Lett. 579, 5822–5829.

    Article  PubMed  CAS  Google Scholar 

  15. Sui G., Soohoo C., El Bashir A., Gay F., Shi Y., Forrester W.C., Shi Y. 2002. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc. Natl. Acad. Sci. USA. 99, 5515–5520.

    Article  PubMed  CAS  Google Scholar 

  16. Paddison P.J., Caudy A.A., Bernstein E., Hannon G.J., Conclin D.S. 2002. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev. 16, 948–958.

    Article  PubMed  CAS  Google Scholar 

  17. Cullen B.R. 2005. Induction of stable RNA interference in mammalian cells. Gene Therapy. 1–6.

  18. Silva J., Chang K., Hannon G.J., Rivas F.V. 2004. RNA-interference-based functional genomics in mammalian cells: Reverse genetics coming of age. Oncogene. 23, 8401–8409.

    Article  PubMed  CAS  Google Scholar 

  19. Heinonen J.E., Smith C.I.E., Nore B.F. 2002. Silencing of Brutons’s tyrosine kinase (Btk) using short interfering RNA duplexes (siRNA). FEBS Lett. 527, 274–278.

    Article  PubMed  CAS  Google Scholar 

  20. Van Es H.H.G., Arts G.-J. 2005. Biology calls the targets: Combining RNAi and disease biology. Drug Discovery Today. 10, 1385–1391.

    Article  PubMed  CAS  Google Scholar 

  21. Boutros M., Kiger A.A., Armknecht S., Kerr K., Hild M., Koch B., Haas S.A., Paro R., Perrimon N. 2004, Genome-wide RNAi analysis of growth and viability in Drosophila cells. Science. 303, 832–835.

    Article  PubMed  CAS  Google Scholar 

  22. Wheeler D.B., Carpenter A.E., Sabatinin D.M. 2005. Cell microarrays and RNA interference chip away at gene function. Nature Genet. 37, S25–S30.

    Article  PubMed  CAS  Google Scholar 

  23. Ziauddin J., Sabatini D.M. 2001. Microarrays of cells expressing defined cDNAs. Nature. 411, 107–110.

    Article  PubMed  CAS  Google Scholar 

  24. Silva J.M., Mizuno H., Brady A., Lucito R., Hannon G.J. 2004. RNA interference microarrays: High-throughput loss-of-function genetics in mammalian cells. Proc. Natl. Acad. Sci. USA. 101, 6548–6552.

    Article  PubMed  CAS  Google Scholar 

  25. Nguyen T.H., Oberholzer J., Birraux J., Majno P., Morel P., Trono D. 2002. Highly efficient lentiviral vector-mediated transduction of nondividing, fully reimplantable primary hepatocytes. Mol. Therapy. 6, 199–209.

    Article  CAS  Google Scholar 

  26. Pfeifer A., Ikawa M., Dayn Y., Verma I.M. 2002. Transgenesis by lentiviral vectors: Lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos. Proc. Natl. Acad. Sci. USA. 99, 2140–2145.

    Article  PubMed  CAS  Google Scholar 

  27. Lukyanov K.A., Chudakov D.M., Fradkov A.F., Labas Y.A., Matz M.V., Lukyanov S. 2006. Discovery and properties of GFP-like proteins from nonbioluminescent anthozoa. Methods Biochem. Anal. 47, 121–138.

    Article  PubMed  Google Scholar 

  28. Fitchen J.H., Hays E.F., Ferrone S. 1982. Expression of the H-2 antigenic complex on murine haematopoietic stem cells. Scand. J. Immunol. 15, 547–551.

    PubMed  CAS  Google Scholar 

  29. Razorenova O.V., Agapova L.S., Budanov A.V., Ivanov A.V., Strunina S.M., Chumakov P.M. 2005. Retroviral reporter systems for assessing the activity of stress-inducible signal transduction pathways controlled by the p53, HIF-1, and HSF-1 transcription factors. Mol. Biol. 39, 286–293.

    CAS  Google Scholar 

  30. Chen S.-T., Iida A., Guo L., Friedmann T., Yee J.-K. 1996. Generation fo packaging cell lines for pseudotyped retroviral vectors of the G protein of vesicular stomatitis virus by using a modified tetracycline inducible system. Proc. Natl. Acad. Sci. USA. 93, 10057–10062.

    Article  PubMed  CAS  Google Scholar 

  31. Zeng Y., Wagner E.J., Cullen B.R. 2002. Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol. Cell. 9, 1327–1333.

    Article  PubMed  CAS  Google Scholar 

  32. Lagos-Quintana M., Rauhit R., Lendeckel W., Tuschl T. 2001. Identification of novel genes coding for small expressed RNAs. Science. 294, 853–858.

    Article  PubMed  CAS  Google Scholar 

  33. Cullen B.R. 2005. RNAi the natural way. Nature Genet. 37, 1163–1165.

    Article  PubMed  CAS  Google Scholar 

  34. Bauknecht T., See R.H., Shi Y. 1996. A novel C/E BP beta-YY1 complex controls the cell-type-specific activity of the human papillomavirus type 18 upstream regulatory region. J. Virol. 70, 7695–7705.

    PubMed  CAS  Google Scholar 

  35. Kolfschoten I.G.M., van Leeuwen B., Berns K., Mullenders J., Beijesbergen R.L., Bernards R., Voorhoeve P.M., Agami R. 2005. A genetic screen identifies PITX1 as a suppressor of RAS activity and tumorigenicity. Cell. 121, 849–858.

    Article  PubMed  CAS  Google Scholar 

  36. Westbrook T.F., Martin E.S., Schlabach M.R., Leng Y., Liang A.C., Feng B., Zhao J.J., Roberts T.M., Mandel G., Hannon G.J., DePinho R.A., Chin L., Elledge S.J. 2005. A genetic screen for candidate tumor suppressors identifies REST. Cell. 121, 837–848.

    Article  PubMed  CAS  Google Scholar 

  37. Silva J.M., Li M.Z., Chang K., Ge W., Golding M.C., Rickles R.J., Siolas D., Hu G., Paddison P.J., Schlabach M.J., Sheth N., Bradshaw J., Burchard J., Kulkarni A., Cavet G., Sachidanandam R., McCombie W.R., Cleary M.A., Elledge S.J., Hannon G.J. 2005. Second-generation shRNA libraries covering the mouse and human genomes. Nature Genet. 37, 1281–1288.

    PubMed  CAS  Google Scholar 

  38. Berns K., Hijmans E.M., Mullenders J., Brummelkamp T.R., Velds A., Heimerlikx M., Kerkhoven R.M., Madiredjo M., Nijkamp W., Weigelt B., Agami R., Ge W., Cavet G., Linsley P.S., Beijersbergen R.L., Bernards R. 2004. A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature. 428, 431–437.

    Article  PubMed  CAS  Google Scholar 

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Original Russian Text © O.A. Guryanova, M. Makhanov, A.A. Chenchik, P.M. Chumakov, E.I. Frolova, 2006, published in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 3, pp. 448–459.

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Guryanova, O.A., Makhanov, M., Chenchik, A.A. et al. Optimization of a genome-wide disordered lentivector-based short hairpin RNA library. Mol Biol 40, 396–405 (2006). https://doi.org/10.1134/S002689330603006X

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  • DOI: https://doi.org/10.1134/S002689330603006X

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