摘要
为了通过脂质体介导的方法获得高效表达目的基因TLR4的绵羊胎儿成纤维细胞作为供体细胞,以便在胚胎移植前的体外筛选过程中确定优越的转基因供体细胞,从而提高体细胞核移植生产抗病转基因绵羊的效率。本研究通过优化脂质体与质粒载体的比例浓度,进而再去转染原代培养的绵羊胎儿成纤维细胞,经G418筛选,以EGFP(Enhanced Green Fluorescent Protein)作为报告基因,从形态学与分子水平鉴定出已经稳定表达目的基因:Toll样受体4(Toll Like Receptor4,TLR4)基因的细胞系。最终经过筛选与纯化得到3个表达目的基因TLR4的细胞克隆,经RT-PCR与相对荧光定量PCR分析,在第二代转染的细胞中TLR4的表达最高,相对于未转染的绵羊胎儿成纤维细胞升高了9.65倍(P<0.01),并藉此为最终制备抗病转基因羊新品种,从提供稳定表达TLR4基因的转基因供体细胞系角度奠定基础。
In order to use the Liposome-mediated method to obtain high-level expression of TLR4 gene sheep fetal fibroblast cells as competent donor cells, which was flexible for the in-vitro screen progress to make sure the effective donor cells before embryo transplantation. The preparation of competent donor cell was critical for the production efficiency of transgenic sheep by somatic cell nuclear transfer （SCNT）. In this experiment, the ovine fetal fibroblast was transfected by lipofection with the reconstructed vector containing the target TLR4, neo and enhanced green fluorescent protein （EGFP, report gene） genes. Then, they were selected by G418 and detected by EGFP positively. Finally, the stable transgenic ovine fetal fibroblast cell line being over expressed TLR4, was established by identification from both morphological and molecular levels. The results showed that using the optimizing ratio of liposome and the TLR4 vector to go to transfect the fetal sheep fibroblast, then selected and purified, we got 3 different positive clones. Compared the non-transfected fetal sheep cells, the clone of highest expression of TLR4 was transfected in the passage 2, and analyzed by reverse transcription PCR and Real-time fluorescent quantitative PCR, its TLR4 level increased 9.65 times （P〈0.01）. Collectively, these stable express high level TLR4 donor cells would contributed for breeding new transgenie sheep breed with somewhat anti-disease characteristic, and would be beneficial to provide competent transgenie donor cells for the production of transgenic animal.
出处
《中国农学通报》
CSCD
2013年第35期63-68,共6页
Chinese Agricultural Science Bulletin
基金
2012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(2011ZX08008005-003)
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"(IDHT 20130515)
