转拟蜘蛛牵丝蛋白基因新疆美利奴细毛羊成纤维细胞系的建立及初步鉴定  

Establishment and Primary Identification of Transgenic Xinjiang Merino Fine Wool Sheep Fibroblast Cell Line Transfected with Spider Dragline Silk-like Protein Gene

作　　者：房君[1] 孟凡华[1] 王海涛[1] 周欢敏[1] 

机构地区：[1]内蒙古农业大学生命科学学院,内蒙古自治区生物制造重点实验室,内蒙古呼和浩特010018

出　　处：《安徽农业科学》2013年第22期9305-9308,共4页Journal of Anhui Agricultural Sciences

基　　金：国家转基因生物新品种培育重大专项(2009ZX08008-008)

摘　　要：[目的]建立含有拟蜘蛛牵丝蛋白基因的新疆美利奴细毛羊成纤维细胞系。[方法]采用脂质体转染,将表达拟蜘蛛牵丝基因的质粒pKap-EGFP4S转入新疆美利奴细毛羊成纤维细胞,通过G418毒性筛选获得阳性细胞系,并采用PCR、RT-PCR检测重组细胞中的目的基因表达。[结果]对体外分离培养的成纤维细胞观察表明细胞形态均为长梭形、活性良好(细胞生长曲线呈S型)、染色体数目为2n=54;通过G418筛选出共43株单克隆细胞系,经PCR鉴定拟蜘蛛牵丝蛋白基因随机整合的细胞系15株,其中挑选4个阳性细胞系经过RT-PCR检测目的基因已转录为mRNA。[结论]成功建立了拟蜘蛛牵丝蛋白基因的新疆美利奴细毛羊成纤维细胞系,为进一步通过体细胞核移植技术制备被毛含转拟蜘蛛牵丝蛋白基因克隆羊奠定基础。[ Objective] q3ae research aimed to establish the fibroblast cell line of Xinjiang Merino Fine Wool Sheep with transgenic spider drag- line silk like protein gene. [ Method] Using liposome transfection, plasmid pKap-EGFP4S which expressed spider dragline silk-like protein gene was transfected into fibroblasts of Xinjiang Merino fine wool sheep. The positive cell line was obtained by G418 toxicity selection. The expression of target gene in reconstitution cell was detected by PCR and RT-PCR. [ Result] The observation on the fibroblasts isolated in vitro and cultured showed that the morphologies of ceils were long fusiform with good activity and the cell growth curve showed S-shaped type. The chromosome number （2n） was 54. 43 clones were selected b）; G418 screening. PCR identification results showed that spider dragline silk like protein gene was randomly inserted into 15 clones. The target gene has been expressed in 4 positive cell lines by RT-PCR detection. [ Conclusion] The fibro- blast cell line of Xinjiang Merino Fine Wool Sheep with transgenic spider dragline silk like protein gene was successfully established, which laid foundation for producing transfer sheep with spider dragline silk-like protein gene by somatic cell nuclear transfer technology.

关 键 词：拟蜘蛛牵丝蛋白基因 细毛羊成纤维细胞 脂质体转染 鉴定 

分 类 号：S188[农业科学—农业基础科学]