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.
Hitz
Christiane
C
Institute for Developmental Genetics, Helmholtz Zentrum München - German Research Center for Environmental Health, Munich, Germany.
Steuber-Buchberger
Patricia
P
Delic
Sabit
S
Wurst
Wolfgang
W
Kühn
Ralf
R
D000818:Animals; D003001:Cloning, Molecular; D053595:Embryonic Stem Cells / Q000502:physiology; D055785:Gene Knockdown Techniques; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology*
D034741:RNA, Small Interfering