McWhir J, Schnieke A E, Ansell R, Wallace H, Colman A, Scott A R, Kind A J
Roslin Institute, Department of Molecular Biology, Midlothian, UK.
Nat Genet. 1996 Oct;14(2):223-6. doi: 10.1038/ng1096-223.
Embryonic stem (ES) cells enable the engineering of precise modifications to the mouse genome by gene targeting. Although there are reports of cultured cell contributions to chimaeras in golden hamster, rat and pig, definitive ES cell lines which contribute to the germline have not been demonstrated in any species but mouse. Among mouse strains, genetic background strongly affects the efficiency of ES isolation, and almost all ES lines in use are derived from strain 129 (refs 1,4,5) or, less commonly, C57BL/6 (refs 6-8). The CBA strain is refractory to ES isolation and there are no published reports of CBA-derived ES lines. Hence, CBA mice may provide a convenient model of ES isolation in other species. In ES derivation it is critical that the primary explant be cultured for a sufficient time to allow multiplication of ES cell progenitors, yet without allowing extensive differentiation. Thus, differences in ES derivation between mouse strains may reflect differences in the control of ES progenitor cells by other lineages within the embryo. Here we describe a strategy to continuously remove differentiated cells by drug selection, which generates germline competent ES lines from genotypes that are non-permissive in the absence of selection.
胚胎干细胞（ES细胞）可通过基因打靶对小鼠基因组进行精确修饰。虽然有报道称培养的细胞可参与金黄仓鼠、大鼠和猪的嵌合体形成，但除小鼠外，尚未在任何物种中证实存在能产生种系的确定性ES细胞系。在小鼠品系中，遗传背景强烈影响ES细胞分离效率，目前使用的几乎所有ES细胞系都源自129品系（参考文献1、4、5），或较少见的C57BL/6品系（参考文献6 - 8）。CBA品系难以分离出ES细胞，且尚无关于源自CBA的ES细胞系的公开报道。因此，CBA小鼠可能为其他物种的ES细胞分离提供一个便利的模型。在ES细胞的分离过程中，至关重要的是将原代外植体培养足够长的时间，以使ES细胞祖细胞增殖，但又不允许其过度分化。因此，小鼠品系之间ES细胞分离的差异可能反映了胚胎内其他谱系对ES细胞祖细胞控制的差异。在此，我们描述了一种通过药物筛选持续去除分化细胞的策略，该策略可从在无筛选条件下不允许产生ES细胞的基因型中产生具有种系能力的ES细胞系。
