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Methods. 2011 Apr;53(4):430-6. doi: 10.1016/j.ymeth.2010.12.015. Epub 2010 Dec 22.

Constitutive and conditional RNAi transgenesis in mice.

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Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Developmental Genetics, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany.


Gene silencing by RNA interference (RNAi) has become a routine method for extracting function from the mammalian genome. Short hairpin (sh) RNAs expressed from stably integrated vectors mediate RNAi both in cultured cells and mice and present therefore a fast alternative to conventional knockout approaches. We describe three strategies to control gene silencing in mice by shRNA expression that can be applied to any transcript of interest. The strategies include germline and inducible cell type-specific knockdowns, which depending on the molecular switch applied can be either permanent (Cre/loxP) or reversible (tetO/tTA). For reliable expression the shRNAs of interest are knocked into a pre-engineered Rosa26 docking site by recombinase mediated cassette exchange (RMCE). ES cells expressing the shRNA of interest can then be used to generate shRNA transgenic mice. The high efficiency of RMCE in ES cells enables the fast production of knockdown mice for in vivo functional analysis.

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