Gallagher Ed J, Lodge Peter, Ansell Ray, McWhir Jim
Roslin Institute, Roslin, Midlothian ETH 9PS, UK.
Transgenic Res. 2003 Aug;12(4):451-60. doi: 10.1023/a:1024225225302.
The isolation of murine embryonic stem (ES) cells has been almost exclusively from the 129 mouse strain. Other mouse strains, such as CBA, have proven refractory to ES cell isolation by conventional means. We previously reported the isolation of 87.5% CBA ES lines by selective ablation of differentiating cells (McWhir et al., 1996). Here, we report the isolation of ES and EG cells from 94% CBA embryos hemizygous and homozygous for a neomycin-resistance transgene under the transcriptional control of the Oct3/4 promoter (Oct/neo). Since expression of the Oct/neo transgene only confers drug resistance to undifferentiated cells of the inner cell mass, selection results in the ablation of differentiating cells from the culture. The efficiency of ES isolation by selective ablation in homozygotes is twice that in heterozygotes. ES isolation frequency in permissive strain 129 embryos is enhanced by treatment with an inhibitor of the extracellular-signal-regulated kinase (ERK) pathway but this effect is not sufficient to permit ES isolation from the CBA strain.
小鼠胚胎干细胞（ES细胞）几乎都是从129小鼠品系中分离得到的。其他小鼠品系，如CBA，已证明难以通过常规方法分离ES细胞。我们之前报道过通过选择性去除分化细胞从CBA中分离出87.5%的ES细胞系（麦克惠尔等人，1996年）。在此，我们报告从94%的CBA胚胎中分离出ES细胞和EG细胞，这些胚胎对于在Oct3/4启动子（Oct/neo）转录控制下的新霉素抗性转基因是半合子和纯合子。由于Oct/neo转基因的表达仅赋予内细胞团未分化细胞抗药性，选择导致从培养物中去除分化细胞。纯合子中通过选择性去除进行ES细胞分离的效率是杂合子中的两倍。在允许的129品系胚胎中，通过用细胞外信号调节激酶（ERK）途径的抑制剂处理可提高ES细胞分离频率，但这种效果不足以使从CBA品系中分离出ES细胞。
