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RNA Interference by Short Hairpin RNAs Expressed in Vertebrate Cells

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mRNA Processing and Metabolism

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 257))

Abstract

RNA interference (RNAi) is now established as a general method to silence gene expression in a variety of organisms. Double-stranded RNA (dsRNA), when introduced to cells, interferes with the expression of homologous genes, disrupting their normal function. In mammals, transient delivery of synthetic short interfering RNAs (siRNAs), which resemble the processed form of standard double stranded RNAi triggers, is effective in silencing mammalian genes. Issues related to transfer efficiency and duration of the silencing effect, however, restrict the spectrum of the applications of siRNAs in mammals. These shortcomings of siRNAs have been solved by the cellular expression of short hairpin RNAs (shRNAs) from DNA vectors. shRNAs are indistinguishable from siRNAs in terms of efficacy and mechanism but can be produced within cells from standard mammalian expression vectors. In this way, shRNA expression makes possible the creation of continuous cell lines and transgenic animals in which suppression of a target gene is stably maintained by RNAi. As a result, the types of RNAi-based gene function analysis that can be carried out in mammals have been greatly expanded. We describe methods for the construction and transfer of stable shRNA expressing vectors suitable for generating loss of function alleles in mammalian cells in vitro or in vivo.

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References

  1. Bleicher, K. H., Bohm, H. J., Muller, K., and Alanine, A. I. (2003) Hit and lead generation: beyond high-throughput screening. Nat. Rev. Drug Dis. 2, 369–378.

    Article  CAS  Google Scholar 

  2. Chanda, S. K. and Caldwell, J. S. (2003) Fulfilling the promise: drug discovery in the post-genomic era. Drug Dis. Today 8, 168–174.

    Article  CAS  Google Scholar 

  3. Kanehisa, M., and Bork, P. (2003) Bioinformatics in the post-sequence era. Nat. Genet. 33, 305–310.

    Article  PubMed  CAS  Google Scholar 

  4. Voorhoeve, P. M., and Agami, R. (2003) Knockdown stands up. Trends Biotechnol. 21, 2–4.

    Article  PubMed  CAS  Google Scholar 

  5. Shi, Y. (2003) Mammalian RNAi for the masses. Trends Genet. 19, 9–12.

    Article  PubMed  Google Scholar 

  6. Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., and Mello, C. C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–11.

    Article  PubMed  CAS  Google Scholar 

  7. Marcus, P. I. and Sekellick, M. J. (1985) Interferon induction by viruses. XIII. Detection and assay of interferon induction-suppressing particles. Virology 142, 411–415.

    Article  PubMed  CAS  Google Scholar 

  8. Gil, J. and Esteban, M. (2000) Induction of apoptosis by the dsRNA-dependent protein kinase (PKR): mechanism of action. Apoptosis 5, 107–114.

    Article  PubMed  CAS  Google Scholar 

  9. Hannon, G. J. (2002) RNA interference. Nature 418, 244–251.

    Article  PubMed  CAS  Google Scholar 

  10. Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498.

    Article  PubMed  CAS  Google Scholar 

  11. Stein, P., Svoboda, P., Anger, M., and Schultz, R. M. (2003) RNAi: Mammalian oocytes do it without RNA-dependent RNA polymerase. RNA 9, 187–192.

    Article  PubMed  CAS  Google Scholar 

  12. Smith, N. A., Singh, S. P., Wang, M. B., Stoutjesdijk, P. A., Green, A. G., and Waterhouse, P. M. (2000) Total silencing by intron-spliced hairpin RNAs. Nature 407, 319–320.

    Article  PubMed  CAS  Google Scholar 

  13. Tavernarakis, N., Wang, S. L., Dorovkov, M., Ryazanov, A., and Driscoll, M. (2000) Heritable and inducible genetic interference by double-stranded RNA encoded by transgenes. Nat. Genet. 24, 180–3.

    Article  PubMed  CAS  Google Scholar 

  14. Kennerdell, J. R. and Carthew, R. W. (2000) Heritable gene silencing in Drosophila using double-stranded RNA. Nat. Biotechnol. 18, 896–898.

    Article  PubMed  CAS  Google Scholar 

  15. Chuang, C. F. and Meyerowitz, E. M. (2000) Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 97, 4985–4990.

    Article  PubMed  CAS  Google Scholar 

  16. Brummelkamp, T. R., Bernards, R., and Agami, R. (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550–553.

    Article  PubMed  CAS  Google Scholar 

  17. McManus, M. T., Petersen, C. P., Haines, B. B., Chen, J., and Sharp, P. A. (2002) Gene silencing using micro-RNA designed hairpins. RNA 8, 842–850.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  19. Paul, C. P., Good, P. D., Winer, I., and Engelke, D. R. (2002) Effective expression of small interfering RNA in human cells. Nat. Biotechnol. 20, 505–508.

    Article  PubMed  CAS  Google Scholar 

  20. Sui, G., Soohoo, C., Affarel, B., Gay, F., Shi, Y., and Forrester, W. C. (2002) A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc. Natl. Acad. Sci. USA 99, 5515–20.

    Article  PubMed  CAS  Google Scholar 

  21. Yu, J. Y., DeRuiter, S. L., and Turner, D. L. (2002) RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc. Natl. Acad. Sci. USA 99, 6047–6052.

    Article  PubMed  CAS  Google Scholar 

  22. McCaffrey, A. P., Meuse, L., Pham, T. T., Conklin, D. S., Hannon, G. J., and Kay, M. A. (2002) RNA interference in adult mice. Nature 418, 38–39.

    Article  PubMed  CAS  Google Scholar 

  23. Carmell, M. A., Zhang, L., Conklin, D. S., Hannon, G. J., and Rosenquist, T. A. (2003) Germline transmission of RNAi in mice. Nat. Struct. Biol. 10, 91–92.

    Article  PubMed  CAS  Google Scholar 

  24. Hemann, M. T., Fridman, J. S., Zilfou, J. T., Hernando, E., Paddison, P. J., Cordon-Cardo, C., et al. (2003) An epi-allelic series of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo. Nat. Genet. 33, 396–400.

    Article  PubMed  CAS  Google Scholar 

  25. Hasuwa, H., Kaseda, K., Einarsdottir, T., and Okabe, M. (2002) Small interfering RNA and gene silencing in transgenic mice and rats. FEBS Lett. 532, 227–230.

    Article  PubMed  CAS  Google Scholar 

  26. Cherington, V., and Higgins, C. (2001) Using retroviral vectors to express SV40 tumor antigens. Meth. Mol. Biol. 165, 129–150.

    CAS  Google Scholar 

  27. Goomer, R. S., and Kunkel, G. R. (1992) The transcriptional start site for a human U6 small nuclear RNA gene is dictated by a compound promoter element consisting of the PSE and the TATA box. Nucleic Acids Res. 20, 4903–4912.

    Article  PubMed  CAS  Google Scholar 

  28. Yu, J. Y., Taylor, J., DeRuiter, S. L., Vojtek, A. B., and Turner, D. L. (2003) Simultaneous inhibition of GSK3alpha and GSK3beta using hairpin siRNA expression vectors. Mol. Ther. 7, 228–236.

    Article  PubMed  CAS  Google Scholar 

  29. McManus, M. T., Haines, B. B., Dillon, C. P., Whitehurst, C. E., van Parijs, L., Chen, J., and Sharp, P. A. (2002) Small interfering RNA-mediated gene silencing in T lymphocytes. J. Immunol. 169, 5754–5760.

    PubMed  CAS  Google Scholar 

  30. Ge, Q., McManus, M. T., Nguyen, T., Shen, C. H., Sharp, P. A., Eisen, H. N., and Chen, J. (2003) RNA interference of influenza virus production by directly targeting mRNA for degradation and indirectly inhibiting all viral RNA transcription. Proc. Natl. Acad. Sci. USA 100, 2718–2723.

    Article  PubMed  CAS  Google Scholar 

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© 2004 Humana Press Inc., Totowa, NJ

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Hannon, G.J., Conklin, D.S. (2004). RNA Interference by Short Hairpin RNAs Expressed in Vertebrate Cells. In: Schoenberg, D.R. (eds) mRNA Processing and Metabolism. Methods in Molecular Biology™, vol 257. Humana Press. https://doi.org/10.1385/1-59259-750-5:255

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  • DOI: https://doi.org/10.1385/1-59259-750-5:255

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-225-4

  • Online ISBN: 978-1-59259-750-5

  • eBook Packages: Springer Protocols

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