期刊
BIOTECHNIQUES
卷 36, 期 1, 页码 74-+出版社
EATON PUBLISHING CO
DOI: 10.2144/04361ST02
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资金
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R37GM025062, R01GM025062] Funding Source: NIH RePORTER
- NIGMS NIH HHS [GM25062] Funding Source: Medline
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 (approximate 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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