利用脂质体法建立转Toll样受体4基因大尾寒羊胎儿成纤维细胞株的研究  

Establishment of Fetal Large-tailed Han Sheep Fibroblasts Cell Line Transfected with Lipofectamine 2000 Vector Containing TLR4 Gene

作　　者：邢书涵[1] 李方舟[1] 曹玉桃[1] 顾美超[1] 倪和民[1] 刘云海[1] 郭勇[1] 

机构地区：[1]北京农学院动物科学技术学院兽医学(中医药)北京市重点实验室,北京102206

出　　处：《中国畜牧兽医》2016年第5期1125-1132,共8页China Animal Husbandry & Veterinary Medicine

基　　金：2013年度北京市教委北京市属高等学校创新团队建设与教师职业发展计划项目"体细胞转基因克隆肉牛新品系培育与利用"(PXM2013_014207_000067);2014年度国家科技重大专项"转基因生物新品种培育"一级子课题"抗病转基因羊扩繁体系初步建立"研究(2014ZX08008005-003);2013年度国家自然基金面上项目"牛体内外植入前胚胎IFNτ的差异表达及对子宫上皮细胞ISG15和Wnt7a表达的体外诱导"(31272526)

摘　　要：利用脂质体转染法获得转Toll样受体4(Toll-like receptors 4,TLR4)基因的成纤维细胞,将其作为供体细胞,再通过体细胞核移植技术构建重构胚,最终生产出转TLR4基因且性别可控的大尾寒羊。本研究通过原代培养大尾寒羊胎儿皮肤成纤维细胞,并进行SRY基因性别鉴定,利用脂质体转染法转入TLR4基因,然后分别从形态学与分子水平检测TLR4基因的表达,将稳定表达转TLR4的成纤维细胞核移植入绵羊去核卵母细胞中构建重构胚。最终获得5个稳定表达TLR4的阳性细胞克隆,经SRY基因性别鉴定为雌性细胞株,通过实时荧光定量PCR分析,在第4代转染的细胞中TLR4表达量最高,比未转染细胞高出7.4816倍(P<0.01),卵裂的重构胚中有EGFP的表达。试验成功构建出含TLR4基因的重构胚,为今后生产出性别可控的抗病转基因绵羊地方新品种奠定基础。Exploiting the method of liposome transfection to obtain modified Toll-like receptors 4 （TLR4） gene fibroblast cells as competent donor,build reconstructed embryos,which finally produced transfected TLR4 gene and gender controllable Large-tailed Han sheep by somatic cell nuclear transfer technology.In this study,through primary culturing the fetus dermal fibroblasts from Large-tailed Han sheep,to identify the SRY gene gender.Using the method of liposome transfection transferred TLR4 gene into fibroblast cells,then detected whether TLR4 gene expression stably or not in morphology level and molecular level respectively,transplanting fibroblast cells which stably expressed TLR4 gene into oocytes without nucleus from Large-tailed Han sheep for building reconstructed embryos.To harvest 5 positive clones by fibroblast cells with stable expression level of TLR4.According to the identification of the SRY gene gender,the cell lines were female.The analysis of Real-time fluorescent quantitative PCR,the fourth generation of transfected cells had the highest expression level of TLR4,which were higher than control group in 7.4816 times （P＜0.01）.The cleavage reconstructed embryos had stable expression level of EGFP.We succeeded in building reconstructed embryos with TLR4 gene and lay the foundation for producing the disease-resistant and gender controllable transgenic sheep in the future.

关 键 词：大尾寒羊 脂质体转染 TLR4基因 重构胚 

分 类 号：Q813[生物学—生物工程]