广西巴马小型猪miR-122慢病毒表达载体的构建及胚胎水平表达  

Construction and Identification of the Lentivirus Vector Containing Guangxi Bama Mini-pig miR-122 Gene

作　　者：司景磊 李龙[1] 郭晓萍[1] 陈秋明[1] 夏利[1] 申玉建 蒋钦杨[1] 兰干球[1] 梁晶[1] 郭亚芬[1] Si Jinglei Li Long Guo Xiaoping Chen Qiuming Xia Li Shen Yujian Jiang Qinyang Lan Ganqiu Liang Jing Guo Yafen(College of Animal Science and Technology, Guangxi University, Nanning, 53000)

机构地区：[1]广西大学动物科学技术学院,南宁530004

出　　处：《基因组学与应用生物学》2016年第9期2306-2310,共5页Genomics and Applied Biology

基　　金："用TALEN技术敲除ABCA1基因制作广西巴马小型猪动脉粥样硬化模型的研究"(桂科能14123006-4);国家现代农业技术体系广西生猪创新团队(nycytxgxcxtd-03-15);广西科技基础条件平台建设项目课题"不同诱导型广西巴马小型猪2型糖尿病动物模型转录组的研究(14-91-07)"共同资助

摘　　要：本实验的目的在于构建PLV-miR-122慢病毒载体并在293T细胞和猪胚胎细胞中进行验证。以广西巴马小型猪基因组DNA为模板,克隆miR-122前体及部分侧翼序列,利用T4连接酶将其连入PLV-CMV-MCS-EF1载体,构建PLV-miR-122慢病毒载体。在293T细胞中过表达PLV-miR-122,通过胞质注射生产转PLV-miR-122的猪胚胎。双酶切和测序结果证明成功构建了PLV-miR-122重组载体,将重组载体转染293T细胞,经胞质注射后观察到其在猪胚胎早期和囊胚期均有绿色荧光表达。本实验成功构建了猪miR-122慢病毒表达载体,在293T细胞中过表达同时生产出转miR-122的阳性胚胎,为后续研究miR-122的功能奠定基础。The purpose of the study was to construct function in the 293T cells and pig embryonic cells, miR- the lentiviral vector of P LV-miR-122 and confirm its 122 precursor and partial flanking sequences was cloned by PCR using Bama mini-pig genomic DNA, and then miR-122 sequence was inserted into PLV-CMV-MCS-EF1 vector by T4 ligase to construct the lentiviral expression vector of PLV-miR-122. The PLV-miR-122 was transfected into 293T cells and then transgenic cloned porcine embroys were produced by intraplasmic injection. The result showed that PLV-miR-122 was confirmed by restriction endonuclease analysis and DNA sequencing. The miR-122 was over-expressed after transfection into 293T cells and the green fluorescence was observed in the development of early embryo and blastocyst stage. In conclusion, this study successfully constructed miR-122 lentivirus vector and over-expressed in 293T cells, and then miR-122 transgenic cloned porcine embryos were produced. These results laid a foundation for further researching the function of miR-122.

关 键 词：广西巴马小型猪 miR-122基因 慢病毒表达载体 胞质注射 

分 类 号：Q78[生物学—分子生物学]