内蒙古白绒山羊VEGF164基因毛囊特异性表达载体的构建及胎儿成纤维细胞的稳定转染  

Construction of a Hair-Follicle-Cell-Specific Expression Vector of Goat VEGF164 Gene and Its Transfection into Caprine Fetal Fibroblasts Cells Stably

作　　者：鲍文蕾[1] 李彬[1] 侯鑫[1] 刘俊娥[1] 郭旭东[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：《中国农业科学》2011年第22期4756-4762,共7页Scientia Agricultura Sinica

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：【目的】构建绒山羊血管内皮生长因子164(vascular endothelial growth factor 164,VEGF164)基因的毛囊特异表达载体并稳定转染胎儿成纤维细胞,筛选获得稳定表达红色荧光蛋白和毛囊特异表达VEGF164并可用于核移植的转基因细胞克隆。【方法】以pCDsRed2载体为基本骨架将VEGF164基因亚克隆到KAP6-1启动子下游,接续连接红色荧光蛋白表达元件,构建VEGF164基因毛囊特异表达载体pCDsRed2-KV(6.3 kb)。外源表达载体以lipofectamineTM2000介导转染胎儿成纤维细胞,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。【结果】在构建的表达载体pCDsRed2-KV中,VEGF164基因被正确连接在毛囊特异性启动子KAP6-1下游,基因下游按顺序连接CMV启动子和红色荧光蛋白基因;外源KAP6-1启动子和VEGF164基因整合到细胞基因组中。【结论】成功构建稳定表达红色荧光蛋白和毛囊特异表达VEGF164的真核表达载体,稳定转染绒山羊胎儿成纤维细胞,为下一步通过克隆技术获得转VEGF164基因绒山羊提供了条件。[Objective] The present study aims at constructing a eukaryotic expression vector pCDsRed2-KV of goat VEGF 164 （vascular endothelial cell growth factor） gene and then to transfer it into Inner Mongolia Cashmere Goat （Capra hircus） fetal fibroblast （GFb） cells to obtain a transgenic cell clones, which stably expresses red fluorescence and expresses VEGF164 in hair follicle cells specifically. The transgenic cell clones can be used for nuclear transplantation. [Method] pCDsRed2-KV （6.3 kb）, a hair-follicle-specific expression vector of VEGFI64, was constructed by connecting VEGF164 gene to downstream of KAP6-1 promoter, and then inserting the KAP6-1 promoter-VEGF164 gene fragment into the basic vector pCDsRed2, which contains a DsRed expression unit. The Inner Mongolia Cashmere Goat fetal fibroblast （GFb） cells were transfected with the expression vector by lipofectamineTM2000. Transgenic cell clones were obtained after screening by G418. The recombinant of exogenous DNA was identified by polymerase chain reaction. [ Result] The sequencing result showed that the VEGF 164 gene was connected properly to the downstream of pKAP6-1, then the CMV promoter and the DsRed2 gene in sequence. Exogenous DNA in the cell clones was examined by PCR and the promoter KAP6-1 as well as VEGFI64 gene has been integrated into GFb cells genome stably. [ Conclusion ] A hair-follicle-cell-specific expression vector of VEGF164 gene was constructed successfully and transfered into GFb cells. These data provide a way to obtain the transgenic goat by nuclear transfer in the future.

关 键 词：内蒙古白绒山羊 VEGF 毛囊特异性表达载体 稳定转染 

分 类 号：S827[农业科学—畜牧学]