Efficient gene knockdowns in mouse embryonic stem cells using microRNA-based shRNAs.
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.
Wang
Jianlong
J
Department of Developmental and Regenerative Biology, Black Family Stem Cell Institute, Mount Sinai School of Medicine, New York, NY, USA.
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000378:metabolism*; D005434:Flow Cytometry; D055785:Gene Knockdown Techniques / Q000379:methods*; D051379:Mice; D035683:MicroRNAs / Q000235:genetics*; D008957:Models, Genetic; D016133:Polymerase Chain Reaction; D034622:RNA Interference / Q000502:physiology*
D035683:MicroRNAs