敲除猪α1,3-半乳糖基转移酶基因并敲入HLA-G1基因的打靶载体构建  

Construction of Targeting Vecter for HLA-G1 Gene Knock-in and α1,3-galactosyltransferase Gene Knock-out

作　　者：周健[1] 林爱星[1] 陈永福[1] 

机构地区：[1]中国农业大学生物技术国家重点实验室

出　　处：《中国生物医学工程学报》2005年第4期498-502,共5页Chinese Journal of Biomedical Engineering

基　　金：国家自然科学基金资助项目(30370726)资助;863计划(2001AA206311)。

摘　　要：本实验室已克隆到猪α1,3半乳糖基转移酶基因(α1,3GT,第九外显子不全)。其中pMD3arm载体上有5.0kb的片段,该片段包括了小部分第8外显子,完整的第8内含子和一部分第9外显子。本实验中利用LAPCR的方法,从猪的基因组中获得约2.0kb的猪α1,3GT的第9外显子序列的扩增产物,将片段克隆于pGEMTeasy载体。将5.0kb和2.0kb片段分别作为打靶载体的5′,3′同源臂,以neo为正选择标记基因,以GFP为负选择标记基因,构建敲除猪α1,3半乳糖基转移酶基因并同时能表达人白细胞抗原HLAG1基因的的打靶载体pZJ。经酶切图谱和测序结果的鉴定成功地构建出敲除猪α1,3半乳糖基转移酶基因并同时能表达人白细胞抗原HLAG1基因的正负双向选择的打靶载体,为异种器官移植提供了很好的探索。We have previously cloned porcine α1,3- galactosyltransferase genomic gene, which includes parts of the exon Ⅷ , the whole intron Ⅷ and part of the exon IX. In this experiment, we have cloned the segment of 2.0kb exon IX from porcine genome by LA-PCR method, which was cloned into pGEM-Teasy vector and identified by DNA sequencing. By taking the segments of 5.0kb and 2.0kb as 5＇and 3＇ homologous targeting arm respectively, the plasmid pGEM-5zf as basic vector, the neo gene as positive selected mark ,the GFP gene as negative selected mark, we constructed the gene targeting vector pZJ according to the strategy of positive-negative selection（ PNS）, in which α1 ,3- galactosyltransferase gene was expected to be knocked out and HLA-G1 gene was expected to be expvessed. The gene targeting vector was identified by enzymic digestion and DNA sequencing. The research work provided an exploration for Xenotransplantation.

关 键 词：α1 3-半乳糖基转移酶基因 基因打靶 人白细胞抗原HLA-G1 

分 类 号：R318[医药卫生—生物医学工程]