绿色荧光蛋白在α-1,3半乳糖基转移酶敲除猪组织器官的表达分析

doi:10.16288/j.yczz.15-160

遗传 ›› 2015, Vol. 37 ›› Issue (12): 1211-1217.doi: 10.16288/j.yczz.15-160

绿色荧光蛋白在α-1,3半乳糖基转移酶敲除猪组织器官的表达分析

李智方^{1, 2}, 冯冲², 纪慧丽^{1, 2}, 石宁宁², 宋小凤², 赵勤丽², 龙川², 潘登科², 杨小淦¹

1. 广西大学,亚热带农业生物资源保护与利用国家重点实验室,南宁 530004;
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:2015-04-15 出版日期:2015-12-20 发布日期:2015-08-12
通讯作者: 杨小淦,博士,副研究员;研究方向：动物繁殖生物技术。E-mail:xgyang@gxu.edu.cn;潘登科,博士,副研究员;研究方向：转基因猪模型。E-mail:pandengke2002@163.com E-mail:LIZHIFANG31@163.com
作者简介:李智方,硕士研究生;专业方向：动物遗传育种与繁殖。E-mail：LIZHIFANG31@163.com
基金资助:
亚热带农业生物资源保护利用国家重点实验室开放课题（编号：SKL201404）和国家高技术研究发展计划（863计划）项目（编号：2012AA020601）资助

Expression analysis of green fluorescent protein in tissues and organs in α-1,3 galactosyltransferase knockout pigs

Zhifang Li^{1, 2}, Chong Feng², Huili Ji^{1, 2}, Ningning Shi², Xiaofeng Song², Qinli Zhao², Chuan Long², Dengke Pan², Xiaogan Yang¹

1. State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources,Guangxi University, Nanning 530004,China;
2. Key Laboratory of Farm Animal Genetic Resource and Germplasm Innovation of Ministry of Agriculture, Institute of Animal Science, Chinese Academy of Agricultural Sciences,Beijing 100193, China

Received:2015-04-15 Online:2015-12-20 Published:2015-08-12

摘要/Abstract

摘要： 猪是人类异种器官移植的理想供体,然而猪-人的异种器官移植会产生剧烈的排斥反应。虽然已制备的α-1,3半乳糖基转移酶基因敲除（Galactosyltransferase gene knockout, GTKO）猪可有效缓解猪-人异种器官移植引起的超急性免疫排斥,但缺少报告基因直观示踪移植后的细胞迁移及器官排斥状态。本文将CAG启动子驱动增强型绿色荧光蛋白（Enhanced green fluorescent protein, EGFP）的表达载体导入GTKO猪耳成纤维细胞,通过体细胞核移植技术制备了EGFP猪。利用双荧光蛋白观测镜、荧光显微镜及定量PCR扩增观察、检测和分析克隆猪各组织器官中EGFP蛋白和转录本的表达状况。结果显示,EGFP蛋白及转录本在克隆猪各组织器官中均有表达,但在肝脏和中枢神经系统中表达较弱。本文成功获得了各组织器官表达EGFP的GTKO猪,为EGFP示踪异种细胞组织移植奠定了基础。

关键词: GTKO, 增强型绿色荧光蛋白, 猪, 异种移植

Abstract: The pig is an ideal source to provide organs because its organ size and physiology are similar to humans. However, an acute rejection will ensue after pig-to-human xenotransplantation. The α-1,3 galactosyltransferase gene knockout (GTKO) pigs were generated in recent years, and could solve the problem of hyperacute rejection. But due to lack of reporting genes, the rejection status of cells and organs post pig-to-human xenotransplantation cannot be visualized. In this study, we introduced the enhanced green fluorescent protein (EGFP) gene driven by the CAG promoter into GTKO porcine ear fibroblasts. Then we produced transgenic pigs expressing the EGFP gene by nuclear transfer technology. Expression levels of EGFP in different tissues and organs of the cloned pig were investigated by Nightsea DFP-1 Fluorescent Protein Flashlight, fluorescence microscope and quantitative PCR assays. The results showed that the protein and transcript of EGFP were expressed in all tissues and organs of the GTKO pig, but the expression was weak in the liver and central nervous system. In conclusion, we have successfully produced the transgenic GTKO pigs expressing EGFP in all tested tissues and organs, which builds up a good basis to track transplanted cells or tissues.

Key words: GTKO, EGFP, pig, xenotransplation

李智方, 冯冲, 纪慧丽, 石宁宁, 宋小凤, 赵勤丽, 龙川, 潘登科, 杨小淦. 绿色荧光蛋白在α-1,3半乳糖基转移酶敲除猪组织器官的表达分析[J]. 遗传, 2015, 37(12): 1211-1217.

Zhifang Li, Chong Feng, Huili Ji, Ningning Shi, Xiaofeng Song, Qinli Zhao, Chuan Long, Dengke Pan, Xiaogan Yang. Expression analysis of green fluorescent protein in tissues and organs in α-1,3 galactosyltransferase knockout pigs[J]. HEREDITAS(Beijing), 2015, 37(12): 1211-1217.

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绿色荧光蛋白在α-1,3半乳糖基转移酶敲除猪组织器官的表达分析

Expression analysis of green fluorescent protein in tissues and organs in α-1,3 galactosyltransferase knockout pigs

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