摘要
为了应用基因重组技术,构建小鼠FAM134B基因慢病毒超表达和短发夹(shRNA)载体,试验根据Gen Bank上登录的小鼠FAM134B基因序列设计引物,扩增该基因开放阅读框(ORF)序列,并克隆到酶切的FUGW载体,同时设计3对FAM134B-siRNA序列克隆到酶切的PsicoR载体,将获得的重组质粒与包装质粒(PSPA和PMD2G)共转染293T细胞包装成病毒颗粒,感染体外培养的3T3-L1前体脂肪细胞,实时荧光定量PCR(q PCR)检测FAM134B基因的超表达及干扰效果。结果表明:重组质粒FUGW-PGC-1α-GFP及PsicoR-FAM134B-GFP测序验证成功,包装后感染3T3-L1前体脂肪细胞发现该基因被成功超表达和干扰。说明成功构建小鼠FAM134B慢病毒超表达和干扰载体,可为该基因在脂代谢中的作用提供重要的工具。
To construct the lentiviral over - expression vector and short hairpin RNA vector of mouse FAM134B gene using gene recombination technology, the primers were designed based on the mouse FAM134B geue sequence registered in GenBank. The open reading frame （ORF） sequence of FAM134B gene was amplified and cloned into the digested FUGW vector. Meanwhile, 3 pairs of sequences for FAM134B - siRNA were designed and cloned into PsicoR vector. The recombinant plasmids and the packed plasmids of PSPA and PMD2G were co - transfected in- to 293T cells and packaged into virus particles to infect 313 - L1 preadipocytes cultured in vitro. Real - time fluorescent quantitative PCR （qPCR） was used to detect the overexpression level of FAM134B gene and the interference effect. The results showed that the sequencing for recombinant plasmids FUGW- PGC -let- GFP and PsicoR -FAM134B -GFP was validated successfully, and it was found that the FAM134B gene was over - expressed and interfered successfully after the lentiviral vector of FAM134B gene was packed to infect the 3T3 - L1 preadipocytes. The result indicates that the lentiviral over - expression vector and interference vector are successfully constructed, and it can provide an important tool for the role of FAM134B gene in the fat metabolism.
出处
《黑龙江畜牧兽医》
CAS
北大核心
2017年第3期5-8,288,共5页
Heilongjiang Animal Science And veterinary Medicine
基金
国家自然科学基金项目(31201990)
四川省应用基础项目(2014JY0080)
