Gene Knockout Protocols pp 101-129
Generation of shRNA Transgenic Mice
RNA interference (RNAi)-mediated gene knockdown has developed into a routine method to assess gene function in cultured mammalian cells in a fast and easy manner. For the use of RNAi in mice, short hairpin (sh) RNAs expressed stably from the genome are a faster alternative to conventional knockout approaches. Here, we describe an advanced strategy for complete or conditional gene knockdown in mice, where the Cre/loxP system is used to activate RNAi in a time- and tissue-dependent manner. Single-copy RNAi constructs are placed into the Rosa26 locus of ES cells by recombinase-mediated cassette exchange and transmitted through the germline of chimaeric mice. The shRNA transgenic offspring can be either directly used for phenotypic analysis or are further crossed to a Cre transgenic strain to activate conditional shRNA vectors. The site-specific insertion of single-copy shRNA vectors allows the expedite and reproducible production of knockdown mice and provides an easy and fast approach to assess gene function in vivo.
Key wordsRNAi transgenic mice Rosa26 Cre/loxP RMCE shRNA
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