中文摘要：

构建了敲除猪α-1,3-GT并敲入人白细胞抗原HLA-G1基因的打靶载体pZJ,其中以新霉素抗性基因（Neo）为正筛选基因,绿色荧光蛋白基因（GFP）为负筛选基因。采用优化的脂质体转染法将打靶载体pZJ转染于一个长势良好的胎猪成纤维细胞系中,经7天G418（600μg/ml）药物筛选,共获得30个Neo抗性细胞克隆,其中12个为非绿色荧光细胞克隆,7个扩大培养良好,提取阳性克隆细胞的基因组DNA并进行PCR检测。经PCR结果检测,打靶载体转入到胎猪成纤维细胞中,其中3个非荧光阳性单克隆细胞在细胞基因组中进行了定点整合。以同样的脂质体转染条件将pZJ转染牛胎儿成纤维细胞,经筛选并对其进行PCR检测,其中2个均为定点整合阳性。该研究为今后克隆出表达HLA-G1而不表达α-1,3-GT基因的个体猪,从而提供可以真正用于临床的异种器官打下了基础,也为猪牛之间的跨物种敲除提供了一个佐证。

英文摘要：

The gene targeting vector pZJ was constructed according to the strategy of positive-negative selection, the Neo gene as the positive selected mark, the GFP gene as the negative selected mark. The pZJ was transferred into pig fetal fibroblast cells using the optimized lipotransfection method. After 7 days＇ G418 selection, 30 neo-positive cell clones were obtained. Among them, 12 were non-green ones, and 7 of which grew well. The genomic DNA samples from the 7 cell clones were identified by PCR method. The gene targeting vector pZJ was transferred into pig fetal fibroblast cells. 3 cell clones were site-specific recombinant by PCR. The pZJ was transferred into bovine fibroblast cells using the same lipotransfection method. After selection, two of the cell clones were identified as the site-specific recombinant by PCR method. The results provide a basis for Xenotransplantation and indicates that α-1,3-GT gene knock-out between species is feasible.