毛囊特异表达载体pCDsR-UI的构建以及绒山羊胎儿成纤维细胞的体外转染和筛选

构建IGF1基因的毛囊特异表达载体,可为今后通过体细胞核移植技术建立转基因克隆绒山羊准备核供体细胞.将绵羊IGF1 cDNA序列,连接到含有UHS启动子序列的克隆载体p19TU的SalI、SphI位点,获得过渡质粒载体p19TUI；pCDsRed2和过渡质粒载体分别经酶切和连接构成表达载体pCDsR-UI.以组织块贴附法分离和培养绒山羊胎儿成纤维细胞,外源性表达载体以脂质体方法转染所培养的第2代成纤维细胞,DMEM/F12+ 10％ FBS、37℃5％CO2中培养,经添加G418筛选,获得了稳定表达红色荧光蛋白的细胞克隆,经特异性PCR鉴定证实外源基因已经整合在细胞基因组中.转基因前后分析细胞生长曲线和染色体核型证明转基因细胞的生长状态良好、各项参数正常.

Construction of a Hair-follicle-cell-specific Expression Vector pCDsR-UI,in vitro Transfection and Screening Vector in Caprine Fetal Fibroblast Cells

Construction of a hair-follicle-cell-specific expression vector of IGF1,is the basis of production of transgenic cashmere goat by nuclear transfer method.Ovine IGF1 cDNA was inserted into p19TU at the sites of SalI and SphI under the control of the mouse ultra high sulfer gene promoter,We obtained the intermediate vecor p19TUI.Then pCDsR-UI was completed by restriction nuclease digestion and ligation of the pCDsRed2 and p19TUI.The caprine fetal fibroblast cells(CFFCs)were isolated by attachment of tissue fragments to the petri dishes.The 2nd passages of CFFCs were transfected with the exogenous expression vector by lipofectamine mediated method.After screening the transfected cells with genetycin in DMEM/F12 plus 10% FBS media at 37℃,5% CO2in a humidified atmosphere,cell colonies which stably expressed red fluorescence were obtained.Identification of the genomic DNA of cell clones by ploymerase chain reaction proved that the exogenous DNA has been integrated into genomes.The growth curves of transgenic cells were drawn and the chromosomal analyses of the cells were performed before and after the transfection.Results showed that the growth of the transgenic cells were prosperous and the parameters of genetics were normal.