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Tissue-specific and cell type–specific RNA interference in vivo

Nature Protocols volume 1, pages 1494–1501 (2006)Cite this article

Abstract

RNA interference (RNAi) is an efficient method for silencing genes in cultured cells. Here we describe a simple RNAi approach for silencing genes in a cell type–specific and tissue-specific way in vivo. The approach, which mimics the means by which naturally occurring 'microRNA's are generated, uses a tissue-specific polymerase II promoter to drive the expression of a short hairpin RNA (shRNA) directed against the gene target. The shRNA is cleaved by ubiquitously expressed endonucleases to form an active small interfering RNA of about 22 nt. As a proof of principle, it has been shown that expression of a shRNA directed against the transcription factor Wilms tumor 1 in transgenic mice reduces that protein specifically in nurse cells in the testis. Our transgenic RNAi approach offers a cost-effective means of rapidly (within months) addressing the function(s) of genes of interest in a wide variety of specific cell types and tissues in mice in vivo.

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Figure 1: The pMan vector.
Figure 2: Generation of an active shRNA.
Figure 3: PCR genotyping of DNA obtained from the tails of mice expressing the shRNA-WT1 transgene17, using transgene-specific primers.
Figure 4

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References

  1. Nagy, A., Gertsenstein, M.V., Vintersten, K. & Behringer, R. In Manipulating the Mouse Embryo: A Laboratory Manual Vol. 1 (eds. Nagy, A., Gertsenstein, M. V., Vintersten, K. & Behringer, R.) 141–161 (Cold Spring Harbor Laboratory Press, New York, 2002).

    Google Scholar 

  2. Le, Y. & Sauer, B. Conditional gene knockout using cre recombinase. Methods Mol. Biol. 136, 477–485 (2000).

    CAS  PubMed  Google Scholar 

  3. Hamilton, A.J. & Baulcombe, D.C. A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286, 950–952 (1999).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  5. Kunath, T. et al. Transgenic RNA interference in ES cell-derived embryos recapitulates a genetic null phenotype. Nat. Biotechnol. 21, 559–561 (2003).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  7. Peng, S., York, J.P. & Zhang, P. A transgenic approach for RNA interference-based genetic screening in mice. Proc. Natl. Acad. Sci. USA 103, 2252–2256 (2006).

    Article  CAS  Google Scholar 

  8. Elbashir, S.M., Lendeckel, W. & Tuschl, T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15, 188–200 (2001).

    Article  CAS  Google Scholar 

  9. Stein, P., Svoboda, P. & Schultz, R.M. Transgenic RNAi in mouse oocytes: a simple and fast approach to study gene function. Dev. Biol. 256, 187–193 (2003).

    Article  CAS  Google Scholar 

  10. Ventura, A. et al. Cre-lox-regulated conditional RNA interference from transgenes. Proc. Natl. Acad. Sci. USA 101, 10380–10385 (2004).

    Article  CAS  Google Scholar 

  11. Tiscornia, G., Tergaonkar, V., Galimi, F. & Verma, I.M. CRE recombinase-inducible RNA interference mediated by lentiviral vectors. Proc. Natl. Acad. Sci. USA 101, 7347–7351 (2004).

    Article  CAS  Google Scholar 

  12. Coumoul, X., Shukla, V., Li, C., Wang, R.H. & Deng, C.X. Conditional knockdown of Fgfr2 in mice using Cre-LoxP induced RNA interference. Nucleic Acids Res. 33, e102 (2005).

    Article  Google Scholar 

  13. Lee, R.C. & Ambros, V. An extensive class of small RNAs in Caenorhabditis elegans. Science 294, 862–864 (2001).

    Article  CAS  Google Scholar 

  14. Zeng, Y. & Cullen, B.R. Structural requirements for pre-microRNA binding and nuclear export by Exportin 5. Nucleic Acids Res. 32, 4776–4785 (2004).

    Article  CAS  Google Scholar 

  15. Zeng, Y. & Cullen, B.R. Sequence requirements for micro RNA processing and function in human cells. RNA 9, 112–123 (2003).

    Article  CAS  Google Scholar 

  16. Lee, N.S. et al. Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat. Biotechnol. 20, 500–505 (2002).

    Article  CAS  Google Scholar 

  17. Rao, M.K. et al. Tissue-specific RNAi reveals that WT1 expression in nurse cells controls germ cell survival and spermatogenesis. Genes Dev. 20, 147–152 (2006).

    Article  CAS  Google Scholar 

  18. Xia, X.G., Zhou, H., Samper, E., Melov, S. & Xu, Z. Pol II-expressed shRNA knocks down Sod2 gene expression and causes phenotypes of the gene knockout in mice. PLoS Genet. 2, e10 (2006).

    Article  Google Scholar 

  19. Dickins, R.A. et al. Probing tumor phenotypes using stable and regulated synthetic microRNA precursors. Nat. Genet. 37, 1289–1295 (2005).

    Article  CAS  Google Scholar 

  20. Chen, X., Wu, J.M., Hornischer, K., Kel, A. & Wingender, E. TiProD: the tissue-specific promoter database. Nucleic Acids Res. 34, D104–D107 (2006).

    Article  CAS  Google Scholar 

  21. Reynolds, A. et al. Rational siRNA design for RNA interference. Nat. Biotechnol. 22, 326–330 (2004).

    Article  CAS  Google Scholar 

  22. Schwarz, D.S. et al. Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199–208 (2003).

    Article  CAS  Google Scholar 

  23. Sambrook, J.R. & Russell, D.W. In Molecular Cloning: A Laboratory Manual Vol. 1 (ed. Irwin, N.) 1.84–1.87 (Cold Spring Harbor Laboratory Press, New York, 2001).

    Google Scholar 

  24. Sambrook, J.R. & Russell, D.W. In Molecular Cloning: A Laboratory Manual Vol. 1 (ed. Irwin, N.) 1.90–1.126 (Cold Spring Harbor Laboratory Press, New York, 2001).

    Google Scholar 

  25. Zhou, H., Xia, X.G. & Xu, Z. An RNA polymerase II construct synthesizes short-hairpin RNA with a quantitative indicator and mediates highly efficient RNAi. Nucleic Acids Res. 33, e62 (2005).

    Article  Google Scholar 

  26. Qian, L. et al. T cell receptor-β mRNA splicing: regulation of unusual splicing intermediates. Mol. Cell. Biol. 13, 1686–1696 (1993).

    Article  CAS  Google Scholar 

  27. Rao, M.K., Wayne, C.M., Meistrich, M.L. & Wilkinson, M.F. Pem homeobox gene promoter sequences that direct transcription in a Sertoli cell-specific, stage-specific, and androgen-dependent manner in the testis in vivo. Mol. Endocrinol. 17, 223–233 (2003).

    Article  CAS  Google Scholar 

  28. Wang, J. & Wilkinson, M.F. Site-directed mutagenesis of large (13-kb) plasmids in a single-PCR procedure. Biotechniques 29, 976–978 (2000).

    Article  CAS  Google Scholar 

  29. Maclean, J.A., II et al. Rhox: a new homeobox gene cluster. Cell 120, 369–382 (2005).

    Article  CAS  Google Scholar 

  30. Zeng, Y., Yi, R. & Cullen, B.R. MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms. Proc. Natl. Acad. Sci. USA 100, 9779–9784 (2003).

    Article  CAS  Google Scholar 

  31. Southern, J. Southern blotting. Nat. Protoc. 1, 518–525 (2006).

    Article  CAS  Google Scholar 

  32. Gudikote, J.P. & Wilkinson, M.F. T-cell receptor sequences that elicit strong down-regulation of premature termination codon-bearing transcripts. EMBO J. 21, 125–134 (2002).

    Article  CAS  Google Scholar 

  33. Miyagishi, M. & Taira, K. U6 promoter-driven siRNAs with four uridine 3′ overhangs efficiently suppress targeted gene expression in mammalian cells. Nat. Biotechnol. 20, 497–500 (2002).

    Article  CAS  Google Scholar 

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Acknowledgements

The authors thank John Pham for technical assistance. This work was supported by National Institutes of Health grants GM58595 and HD042714.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer Center, Houston, 77030, Texas, USA

    Manjeet K Rao & Miles F Wilkinson

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  1. Manjeet K Rao
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  2. Miles F Wilkinson
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Contributions

M.K.R. designed and did the experiments and prepared the manuscript; M.F.W. prepared the manuscript and provided grant support.

Corresponding authors

Correspondence to Manjeet K Rao or Miles F Wilkinson.

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The authors declare no competing financial interests.

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Rao, M., Wilkinson, M. Tissue-specific and cell type–specific RNA interference in vivo. Nat Protoc 1, 1494–1501 (2006). https://doi.org/10.1038/nprot.2006.260

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