作　　者： ; ; ; ; ; ; ;

机构地区： 南方医科大学基础医学院基因工程研究所

出　　处： 《生物技术通讯》 2011年第4期458-462,共5页

摘　　要： 目的:获得α-1,3-半乳糖基转移酶(GGTA1)基因敲除的五指山小型猪胎儿,为异种器官移植研究提供基础平台。方法:以新霉素磷酸转移酶基因(neo)为筛选标记基因构建启动子缺陷型打靶载体,打靶载体线性化后用电转染法将其导入胎儿成纤维细胞中,转染后的细胞经G418筛选后,用PCR法检测药物抗性的细胞克隆,对阳性细胞进行核移植构建重构胚胎及胚胎移植。结果:转染后,经筛选共得到176个具有G418抗性的细胞克隆,经PCR检测,其中2个细胞克隆发生了同源重组;以其中1个GGTA1+/-细胞克隆为供体细胞进行核移植,将重构胚移植到2头自然发情的受体母猪中,1头受体妊娠;第37 d将代孕母猪处死,获得2个胎儿,经PCR和Southern印迹鉴定,均为GGTA1单等位基因敲除胎儿。结论:构建了五指山小型猪GGTA1基因部分外显子4区域敲除的启动子缺陷型打靶载体,获得了GGTA1单等位基因敲除的胎儿,为培育GGTA1基因敲除的五指山小型猪奠定了基础。 Objective： To disrupt GGTA1 gene in Wuzhishan miniature pig fetus and develop a basic platform for xenotransplantation research.Methods： The promoterless gene knockout vector which contained neo positive selection marker was generated.The construct was linearized and electroporated into the porcine fetal fibroblasts cells.The drug resistant cell clones were screened by PCR after G418 selection.Reconstructed embryos established with positive cells were transferred into surrogate sows.Results： G418 resistant cell of 176 clones were screened by PCR,and homologous recombination was found in two cell clones.The GGTA1＋/-cells were used as donors to perform nuclear transplantation.Reconstructed embryos were transferred into two naturally estrous gilts.One early pregnancy was acquired,and the pregnant sow was killed at day 38 and two fetuses were got.The two fetuses which were identified by PCR and Southern blotting were one GGTA1 allelic disrupted.Conclusion： The promoterless vector targeting the exon 4 locus of GGTA1 gene was constructed successfully.One GGTA1 allelic knockout fetus were produced and a basic platform for developing GGTA1 null pigs has been established

关 键 词： 五指山小型猪 基因敲除 基因 胎儿成纤维细胞

领　　域： [生物学]