TY - JOUR
T1 - A Multifunctional Lentiviral-Based Gene Knockdown with Concurrent Rescue that Controls for Off-Target Effects of RNAi
AU - Feng, Yunfeng
AU - Nie, Linghu
AU - Thakur, Meghna Das
AU - Su, Qin
AU - Chi, Zhenfen
AU - Zhao, Yongliang
AU - Longmore, Gregory D.
PY - 2010/12
Y1 - 2010/12
N2 - 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.
AB - 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.
KW - Chemotaxis
KW - Lentivirus
KW - RNAi
KW - ShRNA
KW - α-actinin-1
UR - http://www.scopus.com/inward/record.url?scp=79952284545&partnerID=8YFLogxK
U2 - 10.1016/S1672-0229(10)60025-3
DO - 10.1016/S1672-0229(10)60025-3
M3 - Article
C2 - 21382592
AN - SCOPUS:79952284545
SN - 1672-0229
VL - 8
SP - 238
EP - 245
JO - Genomics, Proteomics and Bioinformatics
JF - Genomics, Proteomics and Bioinformatics
IS - 4
ER -