【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。更多还原

【Abstract】 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.更多还原