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

• Published in: Genomics, proteomics & bioinformatics proteomics & bioinformatics, 2010-12, Vol.8 (4), p.238-245
• Main Authors: Feng, Yunfeng, Nie, Linghu, Thakur, Meghna Das, Su, Qin, Chi, Zhenfen, Zhao, Yongliang, Longmore, Gregory D.
• Format: Article
• Language: English
• Subjects: Bioinformatics; Chemotaxis; Expression vectors; Gene therapy; lentivirus; Lymphocytes T; proteomics; RNA-mediated interference; RNAi; shRNA; siRNA; α-actinin-1; 内部控制; 基因敲除; 多功能; 干扰效应; 慢病毒载体; 绿色荧光蛋白标记; 靶RNA
• ISSN: 1672-0229; 2210-3244
• DOI: 10.1016/S1672-0229(10)60025-3

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.