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 (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.
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Acknowledgments
The author would like to thank Dr. Stuart H. Orkin, an Investigator of Howard Hughes Medical Institute, for his support of the author’s postdoctoral training in his lab when the method was initially developed. The author’s current work is supported by the Seed Fund from the Black Family Stem Cell Institute in Mount Sinai School of Medicine.
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Wang, J. (2010). Efficient Gene Knockdowns in Mouse Embryonic Stem Cells Using MicroRNA-Based shRNAs. In: Zhang, B., Stellwag, E. (eds) RNAi and microRNA-Mediated Gene Regulation in Stem Cells. Methods in Molecular Biology, vol 650. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-769-3_18
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DOI: https://doi.org/10.1007/978-1-60761-769-3_18
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