pEGFP-hTERT真核表达载体的构建及转染牛乳腺细胞方法的优化  

Construction of Eukaryotic Expression Vector pEGFP-hTERT and the Optimization of Scheme on Bovine Mammary Cell Transfection

作　　者：闫晶[1] 贺小英[1] 潘春留[1] 兰志刚[1] 张涌[1] 

机构地区：[1]西北农林科技大学动物医学院生物工程研究所,陕西杨陵712100

出　　处：《动物医学进展》2009年第5期14-19,共6页Progress In Veterinary Medicine

基　　金：国家高技术研究与发展计划(863)项目(2001AA213081)

摘　　要：针对牛乳腺细胞体外培养存活周期短、转染效率低等特点,分别采用Ⅰ型、Ⅱ型胶原酶消化培养法及组织贴壁培养法,比较了牛乳腺细胞的生长、迁移、分化和凋亡等一系列特征。同时构建了携带绿色荧光蛋白报告基因的端粒酶融合表达载体pEGFP-hTERT,采用脂质体分别转染两种真核表达载体pEGFP-hTERT、pEGFP-C1,通过比较载体不同浓度、传代细胞转染适合密度及生长活力、转染后的荧光强度等检测转染效率。结果表明,第3代细胞接种密度大约70%,浓度为350 ng/μL的载体适合于牛乳腺细胞的转染,转染效率大约为25%。试验结果为牛乳腺细胞体外转基因研究奠定了技术基础。Tissue culture, eollagenase Ⅰ and Ⅱ digestion methods of the mammary gland tissue were selected in order to study the traits of bovine mammary epithelial cell＇s growth, differentiation and apoptosis. pEGFP-TERT, one fusion eukaryotic expression vector was constructed with linkage of telomerase reverse transcriptase and green fluorescent protein. Fluorescence intensity was analyzed as a standardization to detect the transfeetion efficiency. Results showed the third passage cells were suit to be transfeeted,with a vector density of 70% and a concentration of 350 ng/μL. The transfection efficiency is 25%. The results will provide efficient strategies for the transgenic study of bovine mammary gland cell in vitro.

关 键 词：牛乳腺上皮细胞 胶原酶 表达载体 转染 

分 类 号：Q291[生物学—细胞生物学] S813.3[农业科学—畜牧学]