Garza Selika, Paik Raehum
Rodent Genome Engineering Core, University of Texas Health San Antonio, San Antonio, TX, USA.
Stanford University, Stanford, CA, USA.
Methods Mol Biol. 2023;2631:253-266. doi: 10.1007/978-1-0716-2990-1_10.
Electroporation of mouse embryos with CRISPR/Cas9 endonuclease tool is a facile and efficient method to edit endogenous genome sequences for generating genetically engineered mouse models (GEMMs). Common genome engineering projects, such as knock-out (KO), conditional knock-out (cKO), point mutation, and small foreign DNA (<1 Kb) knock-in (KI) alleles, can be effectively accomplished with a simple electroporation procedure. The use of electroporation in sequential gene editing at the one-cell (0.7 days post-coitum (dpc)) and at two-cell (1.5 dpc) embryonic stages provides a fast and compelling protocol to safely introduce multiple gene modifications on the same chromosome by limiting chromosomal fractures. In addition, the co-electroporation of the ribonucleoprotein (RNP) complex and single-stranded oligodeoxynucleotide (ssODN) donor DNA with the strand exchange protein Rad51 can significantly increase the number of homozygous founders. Here we describe a comprehensive guideline for mouse embryo electroporation to generate GEMMs and the implementation of Rad51 in RNP/ssODN complex EP medium protocol.
使用CRISPR/Cas9核酸内切酶工具对小鼠胚胎进行电穿孔是一种简便高效的方法，可用于编辑内源基因组序列，以生成基因工程小鼠模型（GEMMs）。常见的基因组工程项目，如基因敲除（KO）、条件性基因敲除（cKO）、点突变以及小的外源DNA（<1 kb）敲入（KI）等位基因，都可以通过简单的电穿孔程序有效完成。在单细胞（合子期后0.7天（dpc））和双细胞（1.5 dpc）胚胎阶段进行连续基因编辑时使用电穿孔，通过限制染色体断裂，提供了一种快速且有说服力的方案，可在同一条染色体上安全引入多个基因修饰。此外，核糖核蛋白（RNP）复合物和单链寡脱氧核苷酸（ssODN）供体DNA与链交换蛋白Rad51的共电穿孔可显著增加纯合子奠基者的数量。在这里，我们描述了一套用于生成GEMMs的小鼠胚胎电穿孔综合指南，以及在RNP/ssODN复合物电穿孔培养基方案中实施Rad51的方法。
