Laboratory of Reproductive Systems Biology, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo;
Research Institute for Microbial Diseases, Osaka University.
J Vis Exp. 2022 Aug 24(186). doi: 10.3791/64385.
The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the efficiency in developing gene-knockout mice by inducing small indel mutation would be sufficient enough, the efficiency of embryo genome editing for making large-size DNA knock-in (KI) is still low. Therefore, in contrast to the direct KI method in embryos, gene targeting using embryonic stem cells (ESCs) followed by embryo injection to develop chimera mice still has several advantages (e.g., high throughput targeting in vitro, multi-allele manipulation, and Cre and flox gene manipulation can be carried out in a short period). In addition, strains with difficult-to-handle embryos in vitro, such as BALB/c, can also be used for ESC targeting. This protocol describes the optimized method for large-size DNA (several kb) KI in ESCs by applying CRISPR/Cas9-mediated genome editing followed by chimera mice production to develop gene-manipulated mouse models.
CRISPR/Cas9 系统通过对受精卵进行直接基因组编辑，使得基因编辑小鼠的研制成为可能。然而，尽管通过诱导小的插入缺失突变来开发基因敲除小鼠的效率已经足够高，但对于进行大尺寸 DNA 敲入（KI）的胚胎基因组编辑效率仍然较低。因此，与胚胎的直接 KI 方法相比，利用胚胎干细胞（ESCs）进行基因靶向，然后通过胚胎注射来开发嵌合体小鼠仍然具有几个优势（例如，体外高通量靶向、多等位基因操作，以及 Cre 和 flox 基因操作可以在短时间内完成）。此外，对于体外处理困难的胚胎，如 BALB/c 等品系，也可以用于 ESC 靶向。本方案描述了通过应用 CRISPR/Cas9 介导的基因组编辑，然后进行嵌合体小鼠的制备，来开发基因操作小鼠模型的 ESCs 中大尺寸 DNA（数 kb）KI 的优化方法。
