IGF-1毛囊特异表达载体的构建以及转染绒山羊胎儿成纤维细胞的研究  

Construction of a Hair-follicle-cell-specific Expression Vector of IGF-1 and Its Transfection into Caprine Fetal Fibroblasts Cells

作　　者：郭旭东[1] 尹俊[2] 杨东山[3] 毛舒燕[1] 宝明涛[1] 旭日干[1] 

机构地区：[1]内蒙古大学哺乳动物生殖生物学及生物技术教育部重点实验室,呼和浩特010021 [2]内蒙古农业大学生物工程学院,呼和浩特010018 [3]中国科学院广州生物医药与健康研究院,广州510663

出　　处：《畜牧兽医学报》2009年第10期1460-1467,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家863计划(2002AA242061);内蒙古自然科学基金(200607010405);内蒙古自治区高等学校科学研究项目(NJ06055)资助

摘　　要：本研究旨在构建绵羊胰岛素样生长因子l(IGF-1)毛囊特异表达载体pCDsR-KI,并转染绒山羊胎儿成纤维细胞,最终获得稳定表达红色荧光并可用于核移植的转基因细胞克隆。通过RT-PCR方法获得IGF-1 cDNA,其与KAP6-1基因启动子片段以及红色荧光蛋白表达元件连接构成IGF-1毛囊特异表达载体pCDsR-KI(大小7.4 kb)。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lipofectamineTM2000介导转染所培养的第2代成纤维细胞,在DMEM/F12+10%FBS、37℃、5%CO2中培养,并添加G418筛选,获得了表达红色荧光蛋白的细胞克隆。经PCR法鉴定证实外源基因已经整合在细胞基因组中。通过分析转基因体细胞的生长曲线和染色体核型证明转基因细胞的生长状态良好、各项参数趋于正常。为下一步通过体细胞核移植技术获得转基因克隆绒山羊准备了核供体细胞。The present study is aimed to construct a hair-follicle-cell-specific expression vector of ovine insulin like growth factor 1 （IGF-1）, transfect it into caprine fetal fibroblast cells （CFF- Cs）, and further to obtain CFFCs cell lines stably expressing red fluorescence, which can be used in nuclear transfer. IGF 1 cDNA was synthesized by reverse transcription polymerase chain reaction. Then pCDsR KI （7.4 kb）, a hair-follicle-cell-specific expression vector of IGF-1, was constructed by connecting with KAP6-1 promoter, as well as a pCDsRed2 expression unit. The CFF- Cs were successfully isolated by attachment of tissue pieces from a fetus of cashmere goat. The 2ne passage of CFFCs were transfected with the expression vector mediated by lipofectamineTM 2000. After screening the transfected cells by genetycin in DMEM/F12 media supplemented with 10% FBS for 12 to 15 days at 37 ℃ in a humidified 5% CO2 condition, cell clones stably expressing red fluorescence were obtained. Identification of the transgene in cell clones by ploymerase chain reaction proved that the exogenous DNA has been integrated into genome. The growth curves of transgenic cells were drawn and the analyses of the cells chromosome were performed. The result showed that growth of the transgenic cells was prosperous and the parameters of genetics were normal. So this study was a preparation of donor cells to obtain transgenic cashmere goats by nuclear transfer.

关 键 词：绒山羊 胰岛素样生长因子l 红色荧光蛋白 毛囊细胞 胎儿成纤维细胞 

分 类 号：S827[农业科学—畜牧学] Q78[农业科学—畜牧兽医]