Site-directed, virus-free, and inducible RNAi in embryonic stem cells.
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.
Wang
Jianlong
J
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, USA.
Theunissen
Thorold W
TW
Orkin
Stuart H
SH
D000818:Animals; D002454:Cell Differentiation; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology*; D005821:Genetic Techniques; D018398:Homeodomain Proteins / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed; D000071317:Nanog Homeobox Protein; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D011995:Recombination, Genetic; D013752:Tetracycline / Q000494:pharmacology
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse; D013752:Tetracycline