Improved silencing vector co-expressing GFP and small hairpin RNA |
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Authors: | Kojima Shin-ichiro Vignjevic Danijela Borisy Gary G |
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Institution: | Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, 303 E. Chicago Ave., Chicago, IL 60611, USA. s-kojima@northwestern.edu |
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Abstract: | Small interfering RNA (siRNA) is a powerful tool for the specific silencing of gene expression. We developed an improved vector, pG-SUPER, that co-expresses green fluorescent protein (GFP) and small hairpin RNA simultaneously to facilitate analysis of silencing at the level of individual cells. As a test system, we analyzed lamin A/C knockdown in HeLa cells. The GFP signal was a reliable reporter (93%-98%) of strong knockdown (approximately 90%) over a wide range of GFP intensities. The GFP reporter made possible the application of fluorescent-activated cell sorting (FACS) to purify the knockdown cell population. Such populations facilitated Western blotting analysis to determine depletion of the target protein. pG-SUPER was also applied to evaluate gene replacement by exogenous genes rendered refractory to siRNA by introducing silent mutations. Recovery of lamin A was linearly correlated to the expression level of the rescue gene. pG-SUPER will expand plasmid-based siRNA applications through the easy and reliable detection of knockdown and rescued cells. |
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