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.
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References
Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001;411:494–8.
Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science 2002;296:550–3.
Lee NS, Dohjima T, Bauer G, et al. Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat Biotechnol 2002;20:500–5.
Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, Conklin DS. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev 2002;16:948–58.
Kuhn R, Streif S, Wurst W. RNA interference in mice. Handb Exp Pharmacol 2007:149–76.
Hasuwa H, Kaseda K, Einarsdottir T, Okabe M. Small interfering RNA and gene silencing in transgenic mice and rats. FEBS Lett 2002;532:227–30.
Rubinson DA, Dillon CP, Kwiatkowski AV, et al. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet 2003;33:401–6.
Lickert H, Takeuchi JK, Von Both I, et al. Baf60c is essential for function of BAF chromatin remodelling complexes in heart development. Nature 2004;432: 107–12.
Hitz C, Wurst W, Kuhn R. Conditional brain-specific knockdown of MAPK using Cre/loxP regulated RNA interference. Nucleic Acids Res 2007;35:e90.
Oberdoerffer P, Kanellopoulou C, Heissmeyer V, et al. Efficiency of RNA interference in the mouse hematopoietic system varies between cell types and developmental stages. Mol Cell Biol 2005;25:3896–905.
Acknowledgements
We thank S. Michailidou, S. Kareth, C. Birke, R. Kneuttinger and A. Tasdemir for excellent technical help and G. Hannon for the pSHAG vector. This work has been funded by the Volkswagen Foundation and the Federal Ministry of Education and Research (BMBF) in the framework of the National Genome Research Network (FKZ:01GR0404).
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Hitz, C., Steuber-Buchberger, P., Delic, S., Wurst, W., Kühn, R. (2009). Generation of shRNA Transgenic Mice. In: Wurst, W., Kühn, R. (eds) Gene Knockout Protocols. Methods in Molecular Biology, vol 530. Humana Press. https://doi.org/10.1007/978-1-59745-471-1_6
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DOI: https://doi.org/10.1007/978-1-59745-471-1_6
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Publisher Name: Humana Press
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Online ISBN: 978-1-59745-471-1
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