DNA Methylation Analysis of Exogenous Genes Transformed into Sheep by Different Methods  

不同方法转基因动物的外源基因甲基化分析(英文)

作　　者：刘莉[1,2] 林嘉鹏[1] 汪立芹[1] 吴阳升[1] 张利[1] 杨楠[1] 蒋香菊[1] 古丽米热[1] 黄俊成[1] 

机构地区：[1]新疆畜牧科学院绵羊中心,新疆乌鲁木齐830000 [2]新疆农业大学,新疆乌鲁木齐830000

出　　处：《Agricultural Science & Technology》2013年第2期197-201,205,共6页农业科学与技术（英文版）

基　　金：Supported by National Natural Science Foundation of China (U1203381);Science and Technology Project of Xinjiang Uygur Autonomous Region (201111113);Science and Technology Support Project of Xinjiang Uygur Autonomous Region (201291147)~~

摘　　要：[Objective] This study aimed to investigate the methylation levels of exogenous genes and promoters and the differences of protein expression in transgenic sheep obtained by different transgenic technologies. [Method] Exogenous genes eGFP （enhanced green fluorescent protein） and FGF5 （fibroblast growth factor 5） were separately transformed into sheep by somatic cell cloning, stem cell cloning and perivitelline injection to obtain transgenic sheep, with CMV as the promoter. Bisulfite sequencing method was adopted to detect the methylation status of the promoter region and coding region of exogenous genes in tail tissues of transgenic sheep. Western blot was adopted to detect the expression level of exogenous genes. [Result] The methylation level of the promoter region with stem cell cloning was the highest, followed by somatic cell cloning, while that with perivitelline injection was the lowest; the methylation level of the eGFP coding region with perivitelline injection was the highest, followed by stem cell cloning; the methylation level of the FGF5 coding region with somatic cell cloning was higher than that with perivitelline injection. The exogenous protein expression level was negatively correlated with the methylation level of the promoter region. [Conclusion] This study indicates that different transgenic methods may influence the methylation level of exogenous genes, thus affecting exogenous gene expression.[目的]检测不同克隆方法所获得的转基因绵羊的外源基因及其启动子的甲基化水平,以及蛋白表达的差异。[方法]通过体细胞克隆、干细胞克隆和卵周隙内注射法将外源基因增强型绿色荧光蛋白(enhanced green fluorescent protein,eGFP)和成纤维细胞内生长因子5(fibroblast growth factor5,FGF5)分别导入绵羊体内,获得转基因绵羊,以CMV为启动子,利用完全重亚硫酸盐沉淀测序法检测绵羊尾部组织外源基因启动子及编码区甲基化状态,Western blot检测外源基因的蛋白表达水平。[结果]启动子区的甲基化水平最高的是干细胞克隆,其次是体细胞克隆,卵周隙注射最低;eGFP编码区的甲基化水平以卵周隙注射最高,其次是干细胞克隆;FGF5编码区甲基化水平体细胞克隆高于卵周隙注射法。外源基因蛋白的表达量同其启动子区域的甲基化水平呈负相关关系。[结论]该研究结果提示不同的克隆方法可能会影响外源基因的甲基化水平,从而影响到外源基因的表达。

关 键 词：DNA methylation Somatic cell cloning Stem cell cloning Perivitelline injection 

分 类 号：S814.8[农业科学—畜牧学]