The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.
Methods Mol Biol. 2023;2637:87-97. doi: 10.1007/978-1-0716-3016-7_7.
The CRISPR/Cas9-mediated genome-editing system enables the development of gene-modified mice using fertilized eggs. However, while the efficiency in developing gene knockout mice by inducing small indel mutations would be good enough, the successful ratio to create large side DNA knock-in (KI) by embryonic genome editing is still low. In contrast to the direct embryo KI method, gene targeting using embryonic stem cells (ESC) followed by chimeric mouse development by blastocyst injection still has several advantages, e.g., high-throughput in vitro targeting/screening or large-size DNA KI such as Cre, CreERT, TetON, and reporter fluorescent protein, or their fusion proteins can be carried out without serving animal lives. The ESC targeting can also be applied to strains such as BALB/c, of which embryos are known to be difficult to handle in vitro. This text describes the optimized method for either short- or large-size DNA KI in ESC by applying CRISPR/Cas9-mediated genome editing followed by chimera mice production to develop gene-manipulated mouse models.
CRISPR/Cas9 介导的基因组编辑系统使使用受精卵来开发基因修饰小鼠成为可能。然而，虽然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好，但通过胚胎基因组编辑成功创建大片段 DNA 敲入 (KI) 的比例仍然较低。与直接胚胎 KI 方法相比，使用胚胎干细胞 (ESC) 进行基因靶向，然后通过囊胚注射进行嵌合体小鼠的开发仍然具有几个优势，例如，高通量的体外靶向/筛选，或者可以进行大片段 DNA KI，如 Cre、CreERT、TetON 和报告荧光蛋白，或它们的融合蛋白，而无需牺牲动物的生命。ESC 靶向也可以应用于 BALB/c 等品系，已知这些品系的胚胎在体外难以处理。本文描述了通过应用 CRISPR/Cas9 介导的基因组编辑，然后通过嵌合体小鼠的产生来开发基因操作小鼠模型，在 ESC 中进行短或大片段 DNA KI 的优化方法。
