A Multifunctional Lentiviral-Based Gene Knockdown with Concurrent Rescue that Controls for Off-Target Effects of RNAi

作     者：Yunfeng Feng Linghu Nie Meghna Das Thakur Qin Su Zhenfen Chi Yongliang Zhao Gregory D. Longmore 

作者机构：Departments of Medicine and Cell Biology Washington University St. Louis MO 63110 USA Key Laboratory of Genome Sciences and Information Cancer Biology and Genetics Group Beijing Institute of Genomics Chinese Academy of Sciences Beijing 100029 China Immunocore Limited Abingdon Oxon OX14 4RX UK 

出 版 物：《Genomics, Proteomics & Bioinformatics》 (基因组蛋白质组与生物信息学报（英文版）)

年 卷 期：2010年第8卷第4期

页      面：238-245页

核心收录：

学科分类：071010[理学-生物化学与分子生物学] 081704[工学-应用化学] 07[理学] 08[工学] 09[农学] 071007[理学-遗传学] 090102[农学-作物遗传育种] 0710[理学-生物学] 1001[医学-基础医学(可授医学、理学学位)] 0817[工学-化学工程与技术] 0714[理学-统计学（可授理学、经济学学位）] 0703[理学-化学] 0901[农学-作物学] 0836[工学-生物工程] 0701[理学-数学] 0812[工学-计算机科学与技术（可授工学、理学学位）] 

主　　题：lentivirus RNAi shRNA α-actinin-1 chemotaxis 

摘      要：The efficient, stable delivery of siRNA into cells, and the appropriate controls for non-specific off-target effects of siRNA are major limitations to functional studies using siRNA technology. To overcome these drawbacks, we have developed a single lentiviral vector that can concurrently deplete endogenous gene expression while expressing an epitope-tagged siRNA-resistant target gene in the same cell. To demonstrate the functional utility of this system, we performed RNAi-depleted α-actinin-1 （α-ACTN1） expression in human T cells. α-ACTN1 RNAi resulted in inhibited chemotaxis to SDF-1α, but it can be completely rescued by concurrent expression of RNAi-resistant α-ACTN1 （rr-α-ACTN1） in the same cell. The presence of a GFP tag on rr-α-ACTN1 allowed for detection of appropriate subcellular localization of rr-α-ACTN1. This system provides not only an internal control for RNAi off-target effects, but also the potential tool for rapid structure-function analyses and gene therapy.