中国农业科学2011，Vol.44Issue(12)：2561-2566,6.DOI:10.3864/j.issn.0578-1752.2011.12.019
转KAP6.1-GFP-蜘蛛拖丝蛋白基因核心序列4S绵羊成纤维细胞株的筛选
Filtration of Transgenic Sheep Skin Fibroblasts with KAP6.1-GFP-polymerized Spider Dragline Silk Protein Gene(4S)
摘要
Abstract
[Objective] This study aims to establish transgenic sheep fibroblast cell line and lay a foundation for transgenic sheep with expression spider dragline silk protein gene in hair follicle by nuclear transplantation. [Method] pcDNA3.1 and pGM-T-KAP6.1(hair follicle-specific promoter) were digested by 5g/Ⅱand Hind Ⅲ, and linked each other. The recombinant plasmid was linked with spider dragline silk protein gene and then linked with pIRES2-EGFP by a series of molecular methods. The eukaryotic expression vector pIRES2-EGFP-4S was constructed. Sheep fibroblasts were transfected with the plasmid by cationic liposome method and G418 was used to filtrate them. After identified, transgenic cell line with spider dragline silk protein gene was established. [Result] The G418 positive cells were detected in vitro. The results showed that cellular morphology was similar with normal fibroblast, cell growth curve was "S" shape, population doubling time (PDT) shorten gradually along with culture time increase, plating efficiency was gradually increasing in 24h. These biological analyses showed the transgenic cells were similar with normal sheep fibroblast. Biological characters of the transgenic cells after freezing-thawing were similar with fresh sheep fibroblast. The PCR result showed that the vector constructed was integrated into sheep genome. [Conclusion] The transgenic cells with polymerized spider dragline silk protein gene were filtrated, and thus laid a foundation for the transgenic sheep with expression spider dragline silk protein in hair follicle.关键词
蜘蛛拖丝蛋白基因/角蛋白启动子/真核表达载体构建/绵羊成纤维细胞/转基因Key words
spider dragline silk protein gene/promoter of sheep keratin/sheep fibroblast/establishment of eukaryotic expression vector/transgene引用本文复制引用
王春生,原璐,宁方勇,吴治昊,朴善花,安铁洙..转KAP6.1-GFP-蜘蛛拖丝蛋白基因核心序列4S绵羊成纤维细胞株的筛选[J].中国农业科学,2011,44(12):2561-2566,6.基金项目
国家自然科学基金资助项目(30771538)、国家转基因生物新品种培育科技重大专项(2009ZX08008-004B) （30771538）
