一种简单的慢病毒载体介导的同时沉默双基因的方法  

A lentivirus-based method for simultaneous silencing of two genes

作　　者：左雨娜 焦德敏[2] 于慧慧[2] 张铸业[2] 李宙阳[2] 袁军[3] 刘爱和[2] 王彦刈[2] 

机构地区：[1]湖南省株洲市一医院血液科,湖南株洲412000 [2]贵州大学生命科学学院,贵州贵阳550025 [3]贵州省人民医院中心实验室,贵州贵阳550000

出　　处：《中国现代医学杂志》2010年第3期369-372,共4页China Journal of Modern Medicine

基　　金：贵州大学研究生创新基金(校研农2009020);贵州省国际科技合作计划(黔科合外G字[2009]700103号);贵州大学人才引进科研项目(贵大人基合字(2007)033号)资助

摘　　要：目的建立一种简单的慢病毒载体介导的同时沉默双基因的方法。方法该研究在慢病毒载体的3'LTR区同时插入沉默两个基因p18和MAD1的shRNA(Small hairpin RNA),通过包装细胞293T和包装质粒包装成假病毒颗粒,感染NIH/3T3细胞。其沉默基因的效果用Western blotting来检测。结果载体构建成功,被感染的NIH3T3细胞中,p18和MAD1基因的蛋白表达被显著抑制。结论该载体构建方法操作简单,无需PCR,及多次连接反应,且可达到两种基因同时被稳定、高效沉默的目的,为相关研究提供了方便。【Objective】To establish a simple method using a single vector to mediate simultaneous silencing of two genes.【Methods】The two synthesized shRNAs（Small hairpin RNA） for silencing of two genes p18 and MAD1 were simultaneously inserted into 3＇LTR of a lentiviral vector.The lentiviruses were obtained by co-transfection of 293T cells with the lentiviral vector and packaging plasmids.The mouse fibroblasts NIH3T3 cells were infected by the lentiviruses.The gene silencing effectiveness was verified by detection of p18 and MAD1 expression using Western blotting.【Results】 The vectors were successfully constructed and the expressions of p18 and MAD1 genes were inhibited effectively in infected NIH3T3 cells.【Conclusion】This method is simple without PCR and many ligation reactions.Using this method,two genes can be simultaneously,stably and efficiently silenced.So,it may provide convenience for some studies.

关 键 词：SHRNA 慢病毒载体 双基因沉默 

分 类 号：Q782[生物学—分子生物学]