Efficient gene knockdowns in mouse embryonic stem cells using microRNA-based shRNAs

Jianlong Wang

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

3 Scopus citations

Abstract

RNA interference (RNAi) is a powerful gene-knockdown technology that has been applied for functional genetic loss-of-function studies in many model eukaryotic systems, including embryonic stem cells (ESCs). Application of RNAi in ESCs allows for dissection of mechanisms by which ESCs self-renew and maintain pluripotency, and also specifying particular cell types needed for cell-replacement therapies. Potent RNAi response can be induced by expression of an microRNA-embedded short-hairpin RNA (shRNA mir) cassette that is integrated in the genome by virus infection or site-specific recombination at a defined locus. In this chapter, I will provide detailed protocols to perform shRNA mir-mediated RNAi studies in mouse ESCs using retrovirus infection and loxP site-directed recombination for efficient constitutive and inducible gene knockdown, respectively.

Original languageEnglish
Title of host publicationRNAi and MicroRNA-Mediated Gene Regulation in Stem Cells
Subtitle of host publicationMethods, Protocols, and Applications
EditorsBaohong Zhang, Edmund Stellwag
Pages241-256
Number of pages16
DOIs
StatePublished - 2010
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume650
ISSN (Print)1064-3745

Keywords

  • RNA interference
  • embryonic stem cells
  • microRNA
  • microRNA-embedded short-hairpin RNA (shRNA )

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