Abstract
RNA interference is a powerful method for suppressing gene expression in mammalian cells. Stable knock-down can be achieved by continuous expression of synthetic short hairpin RNAs, typically from RNA polymerase III promoters. But primary microRNA transcripts, which are endogenous triggers of RNA interference, are normally synthesized by RNA polymerase II. Here we show that RNA polymerase II promoters expressing rationally designed primary microRNA–based short hairpin RNAs produce potent, stable and regulatable gene knock-down in cultured cells and in animals, even when present at a single copy in the genome. Most notably, by tightly regulating Trp53 knock-down using tetracycline-based systems, we show that cultured mouse fibroblasts can be switched between proliferative and senescent states and that tumors induced by Trp53 suppression and cooperating oncogenes regress upon re-expression of Trp53. In practice, this primary microRNA–based short hairpin RNA vector system is markedly similar to cDNA overexpression systems and is a powerful tool for studying gene function in cells and animals.
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References
Brummelkamp, T.R., Bernards, R. & Agami, R. A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550–553 (2002).
Paddison, P.J., Caudy, A.A., Bernstein, E., Hannon, G.J. & Conklin, D.S. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev. 16, 948–958 (2002).
Xia, H., Mao, Q., Paulson, H.L. & Davidson, B.L. siRNA-mediated gene silencing in vitro and in vivo. Nat. Biotechnol. 20, 1006–1010 (2002).
Unwalla, H.J. et al. Negative feedback inhibition of HIV-1 by TAT-inducible expression of siRNA. Nat. Biotechnol. 22, 1573–1578 (2004).
Ling, X. & Li, F. Silencing of antiapoptotic survivin gene by multiple approaches of RNA interference technology. Biotechniques 36, 450–454, 456–460 (2004).
Song, J. et al. Gene silencing in androgen-responsive prostate cancer cells from the tissue-specific prostate-specific antigen promoter. Cancer Res. 64, 7661–7663 (2004).
Zeng, Y., Wagner, E.J. & Cullen, B.R. Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol. Cell 9, 1327–1333 (2002).
Zeng, Y., Cai, X. & Cullen, B.R. Use of RNA polymerase II to transcribe artificial microRNAs. Methods Enzymol. 392, 371–380 (2005).
Boden, D. et al. Enhanced gene silencing of HIV-1 specific siRNA using microRNA designed hairpins. Nucleic Acids Res. 32, 1154–1158 (2004).
Silva, J.M. et al. Second-generation shRNA libraries covering the mouse and human genomes. Nat. Genet. advance online publication xx xxx 2005 (10.1038/ngXXX). [date and doi for hannon].
Hemann, M.T. et al. An epi-allelic series of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo. Nat. Genet. 33, 396–400 (2003).
Lee, Y. et al. MicroRNA genes are transcribed by RNA polymerase II. EMBO J. 23, 4051–4060 (2004).
Cai, X., Hagedorn, C.H. & Cullen, B.R. Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs. RNA 10, 1957–1966 (2004).
Rubinson, D.A. et al. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat. Genet. 33, 401–406 (2003).
Egle, A., Harris, A.W., Bouillet, P. & Cory, S. Bim is a suppressor of Myc-induced mouse B cell leukemia. Proc. Natl. Acad. Sci. USA 101, 6164–6169 (2004).
Schmitt, C.A., McCurrach, M.E., de Stanchina, E., Wallace-Brodeur, R.R. & Lowe, S.W. INK4a/ARF mutations accelerate lymphomagenesis and promote chemoresistance by disabling p53. Genes Dev. 13, 2670–2677 (1999).
van de Wetering, M. et al. Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector. EMBO Rep. 4, 609–615 (2003).
Miyagishi, M., Sumimoto, H., Miyoshi, H., Kawakami, Y. & Taira, K. Optimization of an siRNA-expression system with an improved hairpin and its significant suppressive effects in mammalian cells. J. Gene Med. 6, 715–723 (2004).
Czauderna, F. et al. Inducible shRNA expression for application in a prostate cancer mouse model. Nucleic Acids Res. 31, e127 (2003).
Chen, Y., Stamatoyannopoulos, G. & Song, C.Z. Down-regulation of CXCR4 by inducible small interfering RNA inhibits breast cancer cell invasion in vitro. Cancer Res. 63, 4801–4804 (2003).
Wiznerowicz, M. & Trono, D. Conditional suppression of cellular genes: lentivirus vector-mediated drug-inducible RNA interference. J. Virol. 77, 8957–8961 (2003).
Matsukura, S., Jones, P.A. & Takai, D. Establishment of conditional vectors for hairpin siRNA knockdowns. Nucleic Acids Res. 31, e77 (2003).
Hosono, T. et al. Adenovirus vector-mediated doxycycline-inducible RNA interference. Hum. Gene Ther. 15, 813–819 (2004).
Gupta, S., Schoer, R.A., Egan, J.E., Hannon, G.J. & Mittal, V. Inducible, reversible, and stable RNA interference in mammalian cells. Proc. Natl. Acad. Sci. USA 101, 1927–1932 (2004).
Gossen, M. & Bujard, H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl. Acad. Sci. USA 89, 5547–5551 (1992).
Gossen, M. et al. Transcriptional activation by tetracyclines in mammalian cells. Science 268, 1766–1769 (1995).
Serrano, M., Lin, A.W., McCurrach, M.E., Beach, D. & Lowe, S.W. Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell 88, 593–602 (1997).
Dirac, A.M. & Bernards, R. Reversal of senescence in mouse fibroblasts through lentiviral suppression of p53. J. Biol. Chem. 278, 11731–11734 (2003).
Felsher, D.W. Reversibility of oncogene-induced cancer. Curr. Opin. Genet. Dev. 14, 37–42 (2004).
Harvey, M. et al. In vitro growth characteristics of embryo fibroblasts isolated from p53-deficient mice. Oncogene 8, 2457–2467 (1993).
Stegmeier, F., Hu, G., Rickles, R., Hannon, G.J. & Elledge, S.J. A lentiviral microRNA-based system for single copy Pol II regulated RNAi in mammalian cells. Proc. Natl. Acad. Sci. USA 102, 13212–13217 (2005).
Paddison, P.J. et al. Cloning of short hairpin RNAs for gene knockdown in mammalian cells. Nat. Methods 1, 163–167 (2004).
Schmitt, C.A., Rosenthal, C.T. & Lowe, S.W. Genetic analysis of chemoresistance in primary murine lymphomas. Nat. Med. 6, 1029–1035 (2000).
Schmitt, C.A. et al. A senescence program controlled by p53 and p16INK4a contributes to the outcome of cancer therapy. Cell 109, 335–346 (2002).
Zuker, M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406–3415 (2003).
Acknowledgements
We thank M. Narita for advice on generating vectors; R. Sachidanandam and N. Sheth for shRNA design; A. Malina for the pQTXIX vector; A. Denli, W. Keyes, D. Burgess and A. Bric for experimental assistance; F. Stegmeier and S. Elledge for communicating unpublished results; members of the laboratory of S.W.L. for advice and discussions; and L. Bianco and Cold Spring Harbor Laboratory animal house staff for their assistance. This study was supported by a Mouse Models of Human Cancer Consortium grant and a DNA Tumor Virus grant from the National Cancer Institute. This study was also supported by the Leukemia Research Foundation (R.A.D.), the Helen Hay Whitney Foundation (M.T.H.) and a Ruth L. Kirschstein NRSA (J.T.Z.). D.R.S. is a Beckman Foundation scholar of the Watson School of Biological Sciences. G.J.H. is supported by an Innovator Award from the US Army Breast Cancer Research Program. S.W.L. is an AACR-NCFR Research Professor.
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Dickins, R., Hemann, M., Zilfou, J. et al. Probing tumor phenotypes using stable and regulated synthetic microRNA precursors. Nat Genet 37, 1289–1295 (2005). https://doi.org/10.1038/ng1651
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DOI: https://doi.org/10.1038/ng1651
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