猪miR-486慢病毒表达载体的构建及鉴定  

Construction and Identification of the Lentiviriral Vector Containing the Porcine miR-486 Gene

作　　者：郭晓萍[1] 张笠[1] 黄玥萌 司景磊 鄢胜飞 陈时锦[1] 蒋钦杨[1] 郭亚芬[1] 兰干球[1] 

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》2015年第8期1688-1694,共7页Genomics and Applied Biology

基　　金：国家自然科学基金项目(30960058);国家现代农业产业技术体系广西生猪创新团队项目(nycytxgxcxtd-03-15)共同资助

摘　　要：mi R-486在肌肉生长发育和肌肉疾病的发生中具有重要的功能,本研究旨在构建猪mi R-486慢病毒表达载体,鉴定其在巴马小型猪成纤维细胞中过表达水平,并利用胞质注射生产转基因胚胎,为研究mi R-486在肌肉生长发育中的功能奠定基础。以巴马小型猪基因组DNA为模板,扩增mi R-486前体及部分侧翼序列,利用T4连接酶将其连入p CDH-CMV-MCS-EF1载体,构建LV-mi R-486慢病毒表达载体;在巴马小型猪成纤维细胞中过表达mi R-486并利用Real-time PCR验证其过表达效率,通过胞质注射生产转mi R-486的猪胚胎。双酶切和测序结果证明成功构建了LV-mi R-486重组载体,将慢病毒载体转染巴马小型猪成纤维细胞后mi R-486上调,经胞质注射后在胚胎发育早期和囊胚期均有绿色荧光表达。本研究成功构建了猪mi R-486慢病毒表达载体,在巴马小型猪成纤维细胞中过表达并生产出转mi R-486的阳性胚胎,为后续研究mi R-486的功能奠定基础。mi R-486 plays an important role in muscle development and muscle diseases. The objective of this study was to construct the lentiviral vector of porcine mi R-486 gene and identify the expression level of mi R-486 in Bama mini-pig firbroblast cell, then product transgenic embryos by cytoplasmic injection which would lay a foundation for further research mi R-486 function in muscle growth and development. mi R-486 precursor and partial flanking sequences was amplified by PCR using Bama mini-pig genomic DNA as template, and then mi R-486 sequence was ligated into p CDH-CMV-MCS-EF1 vector by T4 ligase to construct the recombinant plasmid LV-mi R-486. The recombinant plasmid LV-mi R-486 was transfected into Bama mini-pig fibroblast cells and the expression level of mi R-486 was measured by Real-time PCR. Furthermore, mi R-486 transgenic cloned porcine embryos were producted by cytoplasmic injectionion. The result showed that LV-mi R-486 was confirmed by restriction endonuclease analysis and DNA sequencing. The expression level of mi R-486 was up-regulated in Bama mini-pig fibroblast cells after transfection and the green fluorescence was observed in the development of early embryo and blastocyst stage by cytoplasm injection. mi R-486 lentivirus vector was constructed and over-expressed in Bama mini-pig fibroblasts cells successfully, the production of mi R-486 transgenic cloned porcine embryos laid a foundation of the function of mi R-486 in future study.

关 键 词：miR-486 慢病毒表达载体 胞质注射 

分 类 号：Q78[生物学—分子生物学]