Site-directed, Virus-Free, and Inducible RNAi in Embryonic Stem Cells

Proc Natl Acad Sci U S A. 2007 Dec 26;104(52):20850-5. doi: 10.1073/pnas.0710565105. Epub 2007 Dec 18.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Differentiation
  • DNA, Complementary / metabolism
  • DNA-Binding Proteins / metabolism
  • Embryonic Stem Cells / cytology*
  • Genetic Techniques*
  • Homeodomain Proteins / metabolism
  • Kinetics
  • Mice
  • Models, Genetic
  • Mutagenesis, Site-Directed*
  • Nanog Homeobox Protein
  • Plasmids / metabolism
  • Promoter Regions, Genetic
  • RNA Interference*
  • Recombination, Genetic
  • Tetracycline / pharmacology

Substances

  • DNA, Complementary
  • DNA-Binding Proteins
  • Homeodomain Proteins
  • Nanog Homeobox Protein
  • Nanog protein, mouse
  • Tetracycline