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

摘要

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. Theα-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 pro-moter 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 trans-planted cells or tissues.%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。