Kleinhammer Aljoscha, Wurst Wolfgang, Kühn Ralf
Institute for Developmental Genetics, Helmholtz Center Munich, German Research Center for Environmental Health, Neuherberg, Munich, Germany.
Methods Enzymol. 2010;477:387-414. doi: 10.1016/S0076-6879(10)77020-8.
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 from transgenic vectors are a fast alternative to conventional knockout approaches. We describe our strategy to elicit body-wide, cell type-specific, or inducible gene silencing in mice by control of shRNA expression through Cre recombinase or doxycycline. For reproducible expression of shRNAs, vectors are placed into the Rosa26 locus of ES cells by recombinase-mediated cassette exchange and transmitted through the germ line of chimeric mice. The site specific insertion of single copy shRNA vectors allows to expedite and reproducible production of knockdown mice and provides a simple approach to assess gene function in vivo.
RNA干扰（RNAi）介导的基因敲低已发展成为一种快速简便地评估培养的哺乳动物细胞中基因功能的常规方法。对于在小鼠中使用RNAi，从转基因载体表达的短发夹（sh）RNA是传统基因敲除方法的快速替代方法。我们描述了通过Cre重组酶或强力霉素控制shRNA表达，在小鼠中引发全身、细胞类型特异性或诱导性基因沉默的策略。为了实现shRNA的可重复表达，通过重组酶介导的盒式交换将载体导入ES细胞的Rosa26位点，并通过嵌合小鼠的种系进行传递。单拷贝shRNA载体的位点特异性插入允许快速且可重复地产生敲低小鼠，并提供了一种在体内评估基因功能的简单方法。
