Gene Knockout Protocols pp 101-129

Part of the Methods in Molecular Biology book series (MIMB, volume 530)

Generation of shRNA Transgenic Mice

  • Christiane Hitz
  • Patricia Steuber-Buchberger
  • Sabit Delic
  • Wolfgang Wurst
  • Ralf Kühn
Protocol

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 

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Christiane Hitz
    • 1
  • Patricia Steuber-Buchberger
    • 1
  • Sabit Delic
    • 1
  • Wolfgang Wurst
    • 2
  • Ralf Kühn
    • 3
    • 4
  1. 1.Institute for Developmental GeneticsHelmholtz Zentrum München - German Research Center for Environmental HealthMunichGermany
  2. 2.Institute for Developmental GeneticsHelmholtz Zentrum MünchenGermany
  3. 3.Institute for Developmental GeneticsHelmholtz Zentrum München - German Research Center for Environmental HealthMunichGermany
  4. 4.Lehrstuhl für EntwicklungsgenetikTechnische Universität MünchenMunichGermany

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