Department of Medicine, Washington University, St. Louis, MO 63110, USA.
Genomics Proteomics Bioinformatics. 2010 Dec;8(4):238-45. 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-ACTNl) expression in human T cells. α-ACTNl RNAi resulted in inhibited chemotaxis to SDF-lα, but it can be completely rescued by concurrent expression of RNAi-resistant α-ACTNl (rr-α-ACTNl) in the same cell. The presence of a GFP tag on rr-α-ACTNl allowed for detection of appropriate subcellular localization of rr-α-ACTNl. 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.
该系统不仅为 RNAi 脱靶效应提供了内部对照，而且还为快速结构功能分析和基因治疗提供了潜在的工具。
