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

Jianlong Wang

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


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 for specifying particular cell types needed for cell replacement therapies. Potent RNAi response can be induced by expression of a microRNA-embedded short-hairpin RNA (shRNAmir) 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 shRNAmir-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 publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Number of pages14
StatePublished - 2017

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745


  • Embryonic stem cells
  • MicroRNA
  • MicroRNA-embedded short-hairpin RNA (shRNA)
  • RNA interference


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