利用特异性整合酶建立无筛选标记的转LacS基因奶牛细胞系

发布时间：2018-12-18 16:35

【摘要】：转基因动物是生物学研究领域的重要材料,筛选标记基因被广泛应用于转基因动物的生产过程中,然而筛选标记的残留存在生物安全隐患。利用Cre/Loxp重组系统可以实现删除筛选标记基因的目的。本实验构建了pEGFP-N1-LacS载体,通过电转染方法使该载体与体外转录所得的phiC31整合酶mRNA共同转染了奶牛胎儿皮肤成纤维细胞,使LacS基因定点整合入奶牛胎儿皮肤成纤维细胞基因组上,筛选出稳定转染的表达绿色荧光的阳性细胞株。再利用Cre穿膜肽对表达绿色荧光的阳性细胞进行孵育,切除筛选标记基因,获得稳定转染的无绿色荧光的细胞系,为培育无筛选标记基因的转LacS基因克隆奶牛奠定基础。主要结果概括如下：1.以pEGFP-N1为骨架载体,将合成的；attB序列和优化的LacS基因克隆入pEGFP-N1,并在相应位置利用PCR引物添加同方向的两个Loxp序列,构建了真核表达载体p-EGFP-N1-LacS。2.将p-EGFP-N1-LacS质粒和phiC31整合酶mRNA通过电转染的方法共转染奶牛胎儿皮肤成纤维细胞,使目的基因定点整合入奶牛基因组,经过400μg/mL G418筛选出表达绿色荧光的细胞系。3.将pET-28a(-)-His-NLS-TAT-Cre质粒转化BL21(DE3)感受态细胞并诱导表达Cre重组蛋白,经过纯化得到Cre穿膜肽,利用Cre穿膜肽对表达绿色荧光的细胞株进行37-C孵育,切除标记基因。筛选出去除筛选标记基因的无绿色荧光的细胞系,即得到可定点整合并且无筛选标记转LacS的奶牛细胞系。
[Abstract]:Transgenic animals are important materials in the field of biological research. Screening marker genes are widely used in the production process of transgenic animals. Cre/Loxp recombination system can be used to delete the screening marker gene. In this study, pEGFP-N1-LacS vector was constructed and co-transfected into bovine fetal skin fibroblasts by electrotransfection with phiC31 integrase mRNA transcribed in vitro. The LacS gene was integrated into the genome of bovine fetal skin fibroblasts, and a stably transfected positive cell line expressing green fluorescence was selected. The Cre transmembrane peptide was used to incubate the positive cells expressing green fluorescence, and the labeled genes were removed and screened. The stable transfected cell lines without green fluorescence were obtained, which laid the foundation for the cloning of dairy cows with the transgenic LacS gene without screening marker gene. The main results are summarized as follows: 1. Using pEGFP-N1 as skeleton vector, the synthesized attB gene and optimized LacS gene were cloned into pEGFP-N1, and PCR primers were used to add two Loxp sequences in the same direction. The eukaryotic expression vector p-EGFP-N1-LacS.2 was constructed. The p-EGFP-N1-LacS plasmid and phiC31 integrase mRNA were co-transfected into bovine fetal skin fibroblasts by electrotransfection, and the target gene was integrated into the bovine genome. The cell lines expressing green fluorescence were screened by 400 渭 g/mL G418. PET-28a (-)-His-NLS-TAT-Cre plasmid was transformed into BL21 (DE3) competent cells and expressed Cre recombinant protein. Cre transmembrane peptide was purified and incubated with Cre transmembrane peptide for 37-C. Excision of marker gene. The cell lines without green fluorescence were screened out, that is, the cow cell lines which could be integrated at fixed point and transformed into LacS without screening markers were obtained.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823

【相似文献】