Du Yubin, Xie Wen, Zhang Fan, Liu Chengyu
Transgenic Core Facility, Division of Intramural Research, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
Methods Mol Biol. 2019;1874:99-114. doi: 10.1007/978-1-4939-8831-0_6.
The ability to generate chimeric mice through microinjecting embryonic stem (ES) cells into blastocysts is a critical step for the conventional ES cell-mediated knockout technology. In recent years, designer nuclease-based methods, especially the CRISPR technology, have substantially decreased the needs for blastocyst microinjection. However, this method has still remained as a valuable technique for generating sophisticated genetic models as well as for stem cell research. In this chapter, we describe the detailed procedures used in our laboratory on how to use ES cells to produce chimeric mice, including derivation and inactivation of MEF feeder cells, culturing and handling of mouse ES cells, collection and microinjection of blastocysts, and finally implantation of injected blastocysts into the uteri of pseudopregnant surrogate mothers.
通过将胚胎干细胞（ES细胞）显微注射到囊胚中来生成嵌合小鼠的能力是传统ES细胞介导的基因敲除技术的关键步骤。近年来，基于设计核酸酶的方法，尤其是CRISPR技术，已大幅减少了对囊胚显微注射的需求。然而，该方法对于生成复杂的遗传模型以及干细胞研究而言，仍然是一项有价值的技术。在本章中，我们将详细描述我们实验室中使用ES细胞生产嵌合小鼠的具体步骤，包括小鼠胚胎成纤维细胞（MEF）饲养层细胞的来源及失活、小鼠ES细胞的培养与处理、囊胚的收集与显微注射，以及最后将注射后的囊胚植入假孕代孕母亲的子宫。
