Wong Esther S M, Ban Kenneth H, Mutalif Rafidah, Jenkins Nancy A, Copeland Neal G, Stewart Colin L
Institute of Medical Biology, Immunos, Singapore.
Methods Enzymol. 2010;476:265-83. doi: 10.1016/S0076-6879(10)76015-8.
Embryonic stem (ES) cells were first derived from inner cell mass (ICM) explants of preimplantation stage mouse blastocysts some 30 years ago. ES cells are of primary interest as they are used to genetically modify the genome of mice via gene targeting. Although many founder ES lines have been established, there is still a need to obtain new ES lines or their derivatives, often from new mutant mouse lines, to study the function of a mutated gene in different cell types. Existing methods for isolating ES cell lines are inefficient. Here, we describe a reproducible, efficient, and economical method to derive ES cells from different mouse strains using a defined serum-free, serum replacement (KO-SR) media, with 50-85% efficiency. We have derived over 100 ES lines, which when karyotyped>70% were euploid. Two of these lines, when tested, produced germ-line chimeras. We also present procedures for the routine maintenance and karyotyping of the ES cells.
大约30年前，胚胎干细胞（ES细胞）首次从小鼠植入前阶段囊胚的内细胞团（ICM）外植体中分离得到。ES细胞备受关注，因为它们可用于通过基因打靶对小鼠基因组进行基因改造。尽管已经建立了许多起始ES细胞系，但仍需要常常从新的突变小鼠品系中获得新的ES细胞系或其衍生物，以研究突变基因在不同细胞类型中的功能。现有的ES细胞系分离方法效率低下。在此，我们描述了一种可重复、高效且经济的方法，使用限定的无血清、血清替代物（KO-SR）培养基从不同小鼠品系中获得ES细胞，效率为50-85%。我们已经获得了100多个ES细胞系，经核型分析，其中>70%为整倍体。测试的其中两个细胞系产生了种系嵌合体。我们还介绍了ES细胞常规培养和核型分析的方法。
