Wefers Benedikt, Bashir Sanum, Rossius Jana, Wurst Wolfgang, Kühn Ralf
German Center for Neurodegenerative Diseases (DZNE), Feodor-Lynen Str. 17, 81377 Munich, Germany; Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Developmental Genetics, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany.
Max-Delbrück-Centrum für Molekulare Medizin, Robert-Rössle Str. 10, 13125 Berlin, Germany; Berlin Institute of Health, Kapelle-Ufer 2, 10117 Berlin, Germany.
Methods. 2017 May 15;121-122:55-67. doi: 10.1016/j.ymeth.2017.02.008. Epub 2017 Mar 2.
The generation of targeted mouse mutants is a key technology for biomedical research. Using the CRISPR/Cas9 system for induction of targeted double-strand breaks, gene editing can be performed in a single step directly in mouse zygotes. This article covers the design of knockout and knockin alleles, preparation of reagents, microinjection or electroporation of zygotes and the genotyping of pups derived from gene editing projects. In addition we include a section for the control of experimental settings by targeting the Rosa26 locus and PCR based genotyping of blastocysts.
生成靶向小鼠突变体是生物医学研究的一项关键技术。利用CRISPR/Cas9系统诱导靶向双链断裂，可直接在小鼠受精卵中一步完成基因编辑。本文涵盖了敲除和敲入等位基因的设计、试剂制备、受精卵的显微注射或电穿孔以及基因编辑项目所获幼崽的基因分型。此外，我们还设有一个章节，介绍通过靶向Rosa26位点来控制实验设置以及基于PCR的囊胚基因分型。
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