构建LEF1基因毛囊特异表达载体并稳定转染胎儿成纤维细胞  

Construction of Hair Follicle Specific Expression Vector of LEF1 Gene and Its Transfection into Caprine Fetal Fibroblasts Cells Stably

作　　者：李彬[1] 鲍文蕾[1] 张涛[1] 侯鑫[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》2011年第3期141-145,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：旨在构建内蒙古白绒山羊(Capra hircus)淋巴样增强因子-1(Lymphoid enhancer factor,LEF1)基因真核表达载体并转染胎儿成纤维细胞,获得稳定表达红色荧光蛋白及毛囊特异性表达LEF1的转基因细胞克隆。以pCDsRed2载体为基本骨架将LEF1基因亚克隆到KAP6-1启动子下游,连接红色荧光蛋白表达元件,构建LEF1基因毛囊特异表达载体pCDsRed-KL。外源表达载体以lipofectamineTM2000介导转染胎儿成纤维细胞,通过G418筛选获得稳定转染的细胞克隆。PCR鉴定外源基因在细胞基因组中的整合。测序显示构建的表达载体pCDsRed-KL序列中,LEF1基因正确连接在KAP6-1启动子下游,顺序连接CMV启动子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14.0%,经G418筛选得到高效表达红色荧光蛋白转基因细胞克隆。PCR检测显示外源KAP6-1启动子和LEF1基因整合到胎儿成纤维细胞基因组中。The present study aims at constructing a eukaryotic expression vector pCDsRed2-KL of LEF1（Lymphoid enhancer factor 1）gene and then to transfer it into Inner Mongolia goat（Capra hircus）fetal fibroblast（GFb）cells to obtain a transgenic cell line,which stable expresses red fluorescence and hair follicle specific expresses LEF1.The vector pCDsRed-KL was constructed by connecting the LEF1 gene with KAP6-1 promoter and inserting the KAP6-1 promoter-LEF1 fragment into the basic vector pDsRed2,which contains a DsRed expression unit.The GFb cells were transfected with the expression vector by lipofectamineTM2000.Cell clones stably expressing red florescence was obtained after screening by G418.The sequencing result showed the LEF1 gene was connected properly to the downstream of pKAP6-1,then the CMV promoter and the DsRed2 gene in sequence.Identification of the transgene in the cell clones was examined by PCR and the exogenous DNA（pKAP6-1 and LEF1 gene）has been integrated into genome.The stably transfected cell line can express the red fluorescence efficiently.The stable transfection efficiency is about 14%.

关 键 词：内蒙古白绒山羊 淋巴样增强因子-1 毛囊特异性表达载体 稳定转染 

分 类 号：Q78[生物学—分子生物学]