A Multifunctional Lentiviral-Based Gene Knockdown with Concurrent Rescue that Controls for Off-Target Effects of RNAi
10.1016/S1672-0229(10)60025-3
- Author:
Feng YUNFENG
1
;
Nie LINGHU
;
Thakur Das MEGHNA
;
Su QIN
;
Chi ZHENFEN
;
Zhao YONGLIANG
;
D.Longmore GREGORY
Author Information
1. Departments of Medicine and Cell Biology, Washington University, St.Louis, MO 63110, USA
- Keywords:
lentivirus;
RNAi;
shRNA;
α-actinin-1;
chemotaxis
- From:
Genomics, Proteomics & Bioinformatics
2010;08(4):238-245
- CountryChina
- Language:Chinese
-
Abstract:
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.
