临床肝胆病杂志Issue(5)：442-445,4.DOI:10.3969/j.issn.1001-5256.2014.05.015
小鼠肝细胞SMAC基因特异干扰载体的构建及慢病毒包装
Construction of specific interference vector and lentiviral packaging for SMAC gene in mice
吴凡 1易在凤 2田德英1
作者信息
- 1. 华中科技大学同济医学院附属同济医院 感染科，武汉430030
- 2. 华中科技大学同济医学院附属协和医院 中西医结合科，武汉430022
- 折叠
摘要
Abstract
Objective To construct and identify the specific interference vector for second mitochondria -derived activator of caspase (SMAC)gene in mice and perform lentiviral packaging.Methods According to the SMAC gene sequences in mice,three small interfering RNAs (siRNAs)(siRNA1,siRNA2,and siRNA3)and one negative control sequence (siRNAn)were designed and synthesized,and mouse hepatocytes were transfected with the above siRNAs using Lipofectamine 2000.The inhibitory effects of these siRNAs on SMAC mR-NA expression were evaluated by real-time PCR,and the optimal siRNA was screened out accordingly.Oligo DNA was synthesized based on the optimal siRNA and then connected to GV1 15 vector to obtain recombinant interference plasmid.The candidate clones were identified by PCR and sequenced.The recombinant interference plasmid,as well as pHelper 1.0 and pHelper 2.0,was used to transfect 293T cells for lentivirus packaging.Then,cell supernatants were collected and concentrated,and the lentiviral titer was determined by gradient dilution. Comparison between groups was made by analysis of variance,and multiple comparisons were made by SNK-q test.Results The three siRNAs had different inhibitory effects on SMAC mRNA expression in hepatocytes;siRNA1 showed the strongest inhibitory effect,with an inhibition efficiency of 70.3%.Based on siRNA1 ,the oligo DNA was successfully synthesized and correctly connected to the lentiviral vec-tor,as proved by sequencing.The lentiviral titer was 6 ×108 TU/ml,as determined by gradient dilution.Conclusion The recombinant lentivirus that inhibits SMAC expression in mice has been constructed successfully,laying the foundation for further studies on the role of SMAC gene in liver failure.关键词
RNA干扰/SMAC/慢病毒属Key words
RNA interference/SMAC/lentivirus分类
医药卫生引用本文复制引用
吴凡,易在凤,田德英..小鼠肝细胞SMAC基因特异干扰载体的构建及慢病毒包装[J].临床肝胆病杂志,2014,(5):442-445,4.
