Site-directed, virus-free, and inducible RNAi in embryonic stem cells
- *Division of Hematology-Oncology, Children's Hospital and Dana–Farber Cancer Institute, Harvard Stem Cell Institute, and
- ‡Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115
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Contributed by Stuart H. Orkin, November 7, 2007 (received for review September 26, 2007)
Abstract
RNAi is a powerful tool for interrogating gene function in ES cells. Combining the high penetrance of a microRNA-embedded shRNA (shRNA-mir) cassette with a locus-defined, inducible expression strategy, we developed a system for RNAi in mouse ES cells. An shRNA-mir cassette is targeted near the constitutively active HPRT locus under a tetracycline (tet)-regulatable promoter through Cre-mediated site-specific recombination. The major advantage of this system is that the shRNA-mir cassette can be targeted to a precise locus, allowing for control of shRNA-mir expression in an inducible fashion. Induction of an shRNA-mir directed against the pluripotency factor, Nanog, resulted in the loss of self-renewal and differentiation to parietal endoderm-like cells, which can be rescued by the introduction of an RNAi-immune version of Nanog cDNA. Knockdown efficiency can be enhanced by using multiple shRNA-mir hairpins against the target gene, which was further validated by knocking down two additional ES cell factors. This site-directed, virus-free, and tet-inducible RNAi system, designated as SDVFi RNAi in our study, presents an efficient option for controlled gene silencing in ES cells.
Footnotes
- §To whom correspondence should be addressed. E-mail: stuart_orkin{at}dfci.harvard.edu
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Author contributions: J.W. designed research; J.W. and T.W.T. performed research; J.W., T.W.T., and S.H.O. analyzed data; and J.W., T.W.T., and S.H.O. wrote the paper.
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↵ †Present address: Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Cambridge CB2 1QR, United Kingdom.
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The authors declare no conflict of interest.
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Freely available online through the PNAS open access option.
- © 2007 by The National Academy of Sciences of the USA
