Construction of mouse embryonic stem cells for the detection of gene mutation

小鼠胚胎干细胞能在组织培养中保持多能性。胚胎干细胞也可以在体外诱导分化。这些特性使其成为体外研究早期胚胎发生和潜在致畸的极好工具。我们已经在胚胎干细胞中建立了基因报告系统来检测突变。将大肠杆菌黄嘌呤-鸟嘌呤磷酸核糖基转移酶（XPRT）基因（gpt）转染HPRT- ES细胞。在转染gpt基因（pSV2-gpt）后，产生XPRT的存活细胞可以在含有阻断嘌呤核苷酸合成的抑制剂（氨基蝶呤和霉酚酸）的培养基中选择性地以黄嘌呤作为鸟嘌呤核苷酸形成的唯一前体生长。暴露于甲基甲烷磺酸盐（MMS）后，6-TG抗性菌落显著增加。

The mouse ES cells can be maintained in tissue culture with pluripotency. ES cells can also be induced to differentiate in vitro. These properties make it an excellent tool for the study of early embryogenesis and, potentially, teratogenesis in vitro. We have established genetic reporter systems in the ES cells for the detection of mutations. E.coli xanthine-guanine phosporibosyl transferase (XPRT) gene (gpt) was transfected to the HPRT- ES cells. After transfection with the gpt gene (pSV2-gpt), surviving cells producing XPRT can be selectively grown with xanthine as the sole precursor for guanine nucleotide formation in a medium containing inhibitors (aminopterin and mycophenolic acid) that block de novo purinenucleotide synthesis. After methyl methane sulfonate (MMS) exposure, signifcant increases of 6-TG resistant colony were observed.