山羊胎儿成纤维细胞中基因打靶的初步研究

Preliminary Study of Gene Targeting in Goat Fetal Fibroblasts

为评估在山羊胎儿成纤维细胞中通过同源重组进行基因打靶的可行性,构建打靶载体pTarget1,分别以山羊β 酪蛋白基因的5′、3′侧翼区为2条同源臂,中间插入正筛选基因(Neor),外侧插入负筛选基因(HSV tk)。将该载体线性化并纯化后电转染入30日龄的山羊胎儿成纤维细胞中,转染细胞系分别用G418和丙氧鸟苷进行正负药物筛选,存活细胞经PCR检测证明发生了同源重组事件。

To evaluate the feasibility of gene targeting by homologous recombination in goat fetal fibroblasts, a goat β- casein gene targeting vector pTarget1 was constructed. The β- casein gene′s 5′ and 3′ flanking region were used as two homologous arms. The Neo^r gene was inserted between the arms and the HSV-tk gene was flanked the 5′-homologous arm. After linearlization and purification, the targeting vector was introduced into goat fetal fibroblasts derived from 30-day-old fetuses through electroporation. After positive and negative selection by G418 and Ganciclovir respectively, the survival cells were suffered PCR analysis and positive result was detected.