表达红色荧光及转胸腺素β4基因绵羊胎儿成纤维细胞系的制备  

Construction of Transgenic Sheep Fetal Fibroblasts Cell Line Stably Expressing RFP and Thymosin Beta 4

作　　者：王彦凤[1] 梁燕[1] 王玮[1] 史偈君[1] 金永[1] 王晓晶[1] 郭旭东[1] 王潇[1] 王志钢[1] 刘东军[1] 

机构地区：[1]内蒙古大学生命科学学院哺乳动物生殖生物学与生物技术教育部重点实验室,呼和浩特010021

出　　处：《生物技术通报》2010年第12期158-162,共5页Biotechnology Bulletin

基　　金：转基因生物新品种培育重大专项课题研究计划(2008ZX08008-002)

摘　　要：旨在构建胸腺素β4(thymosin beta4,Tβ4)基因真核表达载体并转染绵羊胎儿成纤维细胞,获得稳定表达胸腺素β4及红色荧光蛋白的转基因细胞克隆。将克隆载体pMD19TT中的胸腺素β4基因亚克隆到表达载体pIRES2-DsRed2的多克隆位点,构建表达载体pIRES2-DsRed2-Tβ4,脂质体介导转染绵羊胎儿成纤维细胞,G418筛选获得稳定转染的细胞克隆。RT-PCR检测Tβ4基因在宿主细胞中的转录。测序结果显示,构建的表达载体pIRES2-DsRed2-Tβ4序列中,Tβ4基因正确连接在CMV启动子下游,顺序连接IRES2序列和红色荧光蛋白基因,载体构建正确。脂质体介导的稳定转染效率约为15%,经G418筛选得到转基因细胞克隆并高效表达红色荧光蛋白。RT-PCR检测显示外源Tβ4基因在绵羊胎儿成纤维细胞中得到转录。成功构建具有红色荧光蛋白和新霉素抗性双选择标记的胸腺素β4基因真核表达载体并稳定转染绵羊胎儿成纤维细胞,筛选得到的超表达胸腺素β4绵羊胎儿成纤维细胞系为下一步通过核移植和克隆技术获得转基因绵羊提供了条件。The goat Tβ4（thymosin beta 4）gene was subcloned into a eukaryotic expression vector,and transferred into sheep fetal fibroblast cells to obtain a transgenic cell line which can stably expresses Tβ4 and RFP.The transgenic cell line can be used to nuclear transfer.Tβ4 gene was enzymed from pMD19TT vector and subcloned into multiple cloning sites（MCS）of pIRES2-DsRed2 vector.The pIRES2-DsRed2-Tβ4 vector was constructed.Sheep fetal fibroblast Cells was transfected with the plasmids pIRES2-DsRed2-Tβ4 by liposomes method and screened by G418.Subsequently,the transcription of Tβ4 gene in the transgenic cells was detected by RT-PCR.DNA sequencing showed that Tβ4 gene was inserted between CMV promoter and IRES-DsRed correctly and the eukaryotic expression vector pIRES2-DsRed2-Tβ4 was constructed successfully.Sheep fetal fibroblast cells were transfected with this recombined plasmids and 15% transfect efficiency was obtained.RFP was stably expressed and Tβ4 gene was transcribed in the transgenic cell line after screened with G418.Eukaryotic expression vector pIRES2-DsRed2-Tβ4 was constructed successfully.The goat Tβ4 gene was integrated into sheep fetal fibroblasts genome stably.The obtained cell line was a basis for production of transgenic sheep in the future.

关 键 词：胸腺素Β4 绵羊胎儿成纤维细胞 转基因细胞系 

分 类 号：S826[农业科学—畜牧学]