牛α1,3-半乳糖转移酶基因部分片段的克隆及无启动子打靶载体的构建  

Cloning of the Genomic Fragments of Bovine α 1,3-galactosyltransferase Gene and Construction of a Promotorless Targeting Vector

作　　者：陈立栋[1] 李雪辉[1] 胡宏宇[1] 安晓荣[1] 林爱星[1] 

机构地区：[1]中国农业大学农业生物技术国家重点实验室,北京100094

出　　处：《农业生物技术学报》2003年第6期612-615,共4页Journal of Agricultural Biotechnology

基　　金：国家"863"计划项目(2002AA206311和2001AA213021)。

摘　　要：利用 La-PCR 的方法,从牛的基因组中成功地获得2.4 kb 和5.2 kb 的扩增产物。其中2.4 kb 片段位于5～7外显子之间,包括了完整的第5及第6内含子;5.2 kb 片段位于8～9外显子,包括了完整的第8内含子。2个片段分别克隆于pCR-XL-TOPO 载体。测序结果表明所克隆的两片段均为牛α 1,3半乳糖转移酶基因的基因组序列。2.4 kb 和5.2 kb 的两个片段分别作为打靶载体的5′、3′同源臂构建了无启动子打靶载体 TOPO-IRESneo7.6。电击转染牛胎儿成纤维细胞以期敲除牛的α 1,3半乳糖转移酶基因。Using the LA-PCR method,two fragments 2.4 kb and 5.2 kb were cloned from bovine genome.The 2.4 kb fragment between the fifth exon and the seventh exon included the whole fifth intron and the seventh intron.The 5.2 kb fragment between the eighth exon and the ninth exon included the whole eighth intron.Sequencing result revealed that two fragments were both the segments of bovine α1,3-galactosyltransferase(GT) gene.The 2.4 kb and 5.2 kb fragments were used to construct a promotorless targeting vector TOPO-IRESneo7.6.Electroporating the bovine fetal fibroblast cell with the targeting vector was to knock out bovine GT gene.

关 键 词：牛α1 3-半乳糖转移酶基因 克隆 无启动子打靶载体 生物反应器 

分 类 号：Q78[生物学—分子生物学] S823[农业科学—畜牧学]