家畜生态学报2017，Vol.38Issue(8)：7-13,7.
猪Utx慢病毒过表达/干扰载体的构建和病毒包装
Construction and Package of Porcine Utx LentviralOverexpression/interference Vectors
摘要
Abstract
UTX(ubiquitously transcribed TPR gene on the X chromosome)acts as H3K27me2/3 demethylase and plays important roles in regulating animal developmental processes.According to predicted Sus scrofa Utx gene sequence in the NCBI, complete CDS sequence of Utx was amplified.Subsequently, the pCD513B-CMV-Utx overexpression vector was constructed, and 5 interference shRNA and 1 negative control oligonucleotide fragments was designed by using the acquired Utx complete CDS sequence.The interference shRNAs and negative control oligonucleotide were annealed to form double strand DNA, which were inserted into pCD513B-U6 lentiviral interference vector.The recombination vectors were confirmed by double-enzyme digestion and DNA sequencing.Then interference and overexpression vectors were cotransfected into 293T cell line.The most efficient interference sequence was screened out according to qRT-PCR results.In addition, the 293T cells were transfected by recombined plasmids and three kinds of package plasmids (pRsv-Rev, pGag-Pol, pVSV-G) to package lentivirus.Results showed that the complete Utx gene sequence were successfully amplified.Utx overexpression and interference vectors were successfully constructed.Meanwhile, qRT-PCR results suggested pCD513B-U6-Utx-shRNA-3 was most efficient and the interference efficiency reached 70%.Finally, Lenti-Utx-shRNA, Lenti-NC and Lenti-Utx were successfully packaged, which titers were 6.1×107 TU/mL, 4.8×107 TU/mL and 3.9×105 TU/mL respectively.Taken together, these results make foundation for the funetionel studies of the Utx gene in porcine cells.关键词
组蛋白去甲基化/Utx/shRNA/慢病毒/过表达载体/干扰载体Key words
histone demethylation/Utx/shRNA/lentivirus/overexpression vector/interference vector分类
农业科技引用本文复制引用
闫海龙,陈晓旭,朱晋生,张鹏飞,李爽,曾文先..猪Utx慢病毒过表达/干扰载体的构建和病毒包装[J].家畜生态学报,2017,38(8):7-13,7.基金项目
教育部博士点基金项目(博导类)(20130204110017),国家自然科学基金(面上)(31272439) （博导类）
