The Centre for Phenogenomics, Toronto M5T 3H7, Canada.
The Centre for Phenogenomics, Toronto M5T 3H7, Canada; The Hospital for Sick Children, Toronto M5G 1X8, Canada.
Methods. 2021 Jul;191:32-43. doi: 10.1016/j.ymeth.2021.01.005. Epub 2021 Jan 30.
Knockout mice are used extensively to explore the phenotypic effects of mammalian gene dysfunction. With the application of RNA-guided Cas9 nuclease technology for the production of knockout mouse lines, the time, as well as the resources needed, to progress from identification of a gene of interest to production of a knockout line is significantly reduced. Here we present our standard methodology to produce knockout mouse lines by the electroporation of Cas9 ribonucleoprotein (RNP) into mouse zygotes. Using this protocol, we have obtained an 80% success rate in the generation of founders for null alleles with a subsequent 93% germline transmission rate. These methods rely on equipment already present in the majority of transgenic facilities and should be straightforward to implement where appropriate embryo handling expertise exists.
敲除小鼠被广泛用于探索哺乳动物基因功能障碍的表型效应。随着 RNA 引导的 Cas9 核酸酶技术在敲除小鼠品系生产中的应用，从鉴定感兴趣的基因到生产敲除品系所需的时间和资源大大减少。在这里，我们介绍了通过将 Cas9 核糖核蛋白（RNP）电穿孔到小鼠受精卵中生产敲除小鼠品系的标准方法。使用该方案，我们已经成功获得了 80%的具有随后 93%种系传递率的 null 等位基因的 founder 的成功率。这些方法依赖于大多数转基因设施中已经存在的设备，并且在适当的胚胎处理专业知识存在的情况下应该易于实施。
