Protocol

Gene Knockout Protocols

Volume 530 of the series Methods in Molecular Biology pp 101-129

Date:

Generation of shRNA Transgenic Mice

  • Christiane HitzAffiliated withInstitute for Developmental Genetics, Helmholtz Zentrum München - German Research Center for Environmental Health
  • , Patricia Steuber-BuchbergerAffiliated withInstitute for Developmental Genetics, Helmholtz Zentrum München - German Research Center for Environmental Health
  • , Sabit DelicAffiliated withInstitute for Developmental Genetics, Helmholtz Zentrum München - German Research Center for Environmental Health
  • , Wolfgang WurstAffiliated withInstitute for Developmental Genetics, Helmholtz Zentrum München
  • , Ralf KühnAffiliated withInstitute for Developmental Genetics, Helmholtz Zentrum München - German Research Center for Environmental HealthLehrstuhl für Entwicklungsgenetik, Technische Universität München

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Abstract

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 words

RNAi transgenic mice Rosa26 Cre/loxP RMCE shRNA