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

畜牧兽医学报

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

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*

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

收稿日期:2012-10-22 出版日期:2013-04-23 发布日期:2013-04-23
通讯作者: 潘登科，副研究员，E-mail:pandengke@yahoo.com.cn；帅素容，教授，E-mail:srshuai@sohu.com
作者简介:王飞（1986-），男，四川荥经人，硕士生，主要从事动物转基因与克隆的研究，E-mail: wangfei860820@126.com；冯冲（1981-），男，辽宁沈阳人，博士生，主要从事动物转基因与克隆的研究，E-mail:fengchong81358247@163.com。王飞与冯冲同为第一作者
基金资助:
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）

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

WANG Fei^1,2 , FENG Chong² , LONG Chuan², YUE Cheng-he^1,2, WANG Ning², LI Xi-rui², CAO Sui-zhong¹, LI Ming-zhou¹, CHU Ming-xing², PAN Deng-ke^2*, SHUAI Su-rong^1*

1. Institute of Animal Genetics&Breeding, Sichuan Agricultural University, Ya’an 625014, China; 2. Key Laboratory of Farm Animal Genetic Resources and Germplasm Innovation ofMinistry of Agriculture， Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China

Received:2012-10-22 Online:2013-04-23 Published:2013-04-23

摘要/Abstract

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。

Abstract:

The production of the α 1,3-galactosyltransferase (GGTA1) gene-deficient Wuzhishan miniature pigs will provide the valuable genetically modificated animal models for the further development of the xenotransplantation between the pig and human. Based on our previous, establishment 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 reconstructed with the GGTA1^-/- cells were transferred into 13 recipient pigs, four of them were pregnant, and producing 12 liveborns. Absences of GGTA1 in the cloned pigs were confirmed by PCR and Southern blotting. Flow cytometric analysis revealed that α1,3Gal 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.

中图分类号:

Q812

王飞，冯冲，龙川，岳成鹤，王宁，李西睿，曹随忠，李明洲，储明星，潘登科，帅素容. 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[J]. 畜牧兽医学报, doi: .

WANG Fei,FENG Chong,LONG Chuan, YUE Cheng-he, WANG Ning, LI Xi-rui, CAO Sui-zhong, LI Ming-zhou, CHU Ming-xing, PAN Deng-ke, SHUAI Su-rong. Production of α1,3-galactosyltransferase Gene-Deficient Wuzhishan Miniature Pigs by Fibroblasts Bearing Loss of Heterozygosity Mutations[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, doi: .

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