利用体细胞LOH突变制备α1,3-半乳糖基转移酶基因(GGTA1)缺失的五指山小型猪  

Production of α1,3-galactosyltransferase Gene-Deficient Wuzhishan Miniature Pigs by Fibroblasts Bearing Loss of Heterozygosity Mutations

作　　者：王飞[1,2] 冯冲[2] 龙川[2] 岳成鹤[1,2] 王宁[2] 李西睿[2] 曹随忠[1] 李明洲[1] 储明星[2] 潘登科[2] 帅素容[1] 

机构地区：[1]四川农业大学,动物遗传育种研究所,雅安625014 [2]中国农业科学院北京畜牧兽医研究所,农业部畜禽遗传资源与种质创新重点实验室,北京100193

出　　处：《畜牧兽医学报》2013年第4期522-527,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家重点基础研究发展计划"973计划"(2011CBA01004);中央级公益性科研院所基本科研业务费项目(2011cj-10-2)

摘　　要：制备α1,3-半乳糖基转移酶(αl,3-galactosyltransferase,GGTA1)基因缺失的五指山小型猪,为我国异种器官移植研究奠定基础。在已经获得的GGTA1单等位基因敲除(GGTA+/-)五指山小型猪基础上,建立GGTA1+/-猪耳成纤维细胞系。利用特异性结合α1,3Gal的药物GSI-B4联合免疫磁珠筛选,成功分离出自发杂合性缺失(Loss of heterozygosity,LOH)的双等位基因敲除(GGTA1-/-)耳成纤维细胞。单细胞克隆培养与PCR鉴定后,以GGTA1-/-细胞为核供体,体外成熟的猪卵母细胞为核受体构建克隆胚胎并移植。先后将3 122枚重构胚移植到13头受体母猪,其中4头怀孕,产仔12头。采用PCR和Southern blotting进行GGTA1基因型检测,11头为GGTA1-/-猪;流式细胞术分析表明,GGTA1-/-仔猪耳成纤维细胞不表达α1,3Gal。获得能克服异种器官超急性排斥反应(HAR)的GGTA1-/-猪,不仅为异种器官供体的进一步基因修饰提供了平台,也为异种移植临床前研究提供了宝贵资源。The production of the a 1,3-galactosyltransferase （GGTA1） gene-deficient Wuzhishan miniature pigs will provide the valuable genetically modificated animal models for the further de- velopment of the xenotransplantation between the pig and human. Based on our previous, estab- lishment of the fibroblast cell lines using Wuzhishan miniature pig which been knocked out single GGTA1 allele by the way of promoter-trap, then selected spontaneous loss of heterozygosity cells from fibroblast cultures of heterozygous animals using a biotin-labeled IB4 lectin attached to streptavidin-coated magnetic beads. A total of 3 122 somatic nuclear transferred embryos recon- structed with the GGTA1-/- cells were transferred into 13 recipient pigs, four of them were preg-nant, and producing 12 liveborns. Absences of GGTA1 in the cloned pigs were confirmed by PCR and Southern blotting. Flow cytometric analysis revealed that a1,3Gal antigens were not present in the cells of the cloned GGTA1-/- pigs. The production of GGTA1-/- pigs provided a typical paradigm and an important resource for the further development of genetically modified pigs for make organs tailored for transplantation to humans.

关 键 词：Α1 3-半乳糖基转移酶 自发杂合性缺失 异种器官移植 体细胞核移植 五指山小型猪 

分 类 号：Q812[生物学—生物工程]