Site-directed, virus-free, and inducible RNAi in embryonic stem cells.

Site-directed, virus-free, and inducible RNAi in embryonic stem cells. Research Abstract Details 

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Site-directed, virus-free, and inducible RNAi in embryonic stem cells. Abstract Text:

Jianlong Wang,Thorold W Theunissen,Stuart H Orkin,Jianlong Wang,Thorold W Theunissen,Stuart H Orkin,Jianlong Wang,Thorold W Theunissen,Stuart H Orkin,

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.

Site-directed, virus-free, and inducible RNAi in embryonic stem cells. Publishing Authors By Initials

J Wang,TW Theunissen,SH Orkin,J Wang,TW Theunissen,SH Orkin,J Wang,TW Theunissen,SH Orkin,

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Site-directed, virus-free, and inducible RNAi in embryonic stem cells. Journal Published:

PUBLICATION TYPE: Journal Article

Journal: Proceedings of the National Academy of Sciences of

VOLUME: 104

Page Numbers: 20850-5

Journal Abbreviation: Proc. Natl. Acad. Sci. U.S.A.

ISSN: 1091-6490

DAY: 18

MONTH: 12

YEAR: 2007

Site-directed, virus-free, and inducible RNAi in embryonic stem cells. Information

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LANGUAGE: eng

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Grant and Affiliation Information for Site-directed, virus-free, and inducible RNAi in embryonic stem cells.

AFFILIATION: 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.

Country: United States

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MEDLINETA: Proc Natl Acad Sci U S A

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