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.
作者
司景磊
李龙
郭晓萍
陈秋明
夏利
申玉建
蒋钦杨
兰干球
梁晶
郭亚芬
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)
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
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
