Kojima Shin-ichiro, Vignjevic Danijela, Borisy Gary G
Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, 303 E. Chicago Ave., Chicago, IL 60611, USA.
Biotechniques. 2004 Jan;36(1):74-9. doi: 10.2144/04361ST02.
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.
小干扰RNA（siRNA）是一种用于特异性沉默基因表达的强大工具。我们开发了一种改进的载体pG-SUPER，它能同时共表达绿色荧光蛋白（GFP）和小发夹RNA，以便于在单个细胞水平上分析沉默情况。作为一个测试系统，我们分析了HeLa细胞中核纤层蛋白A/C的敲低情况。在广泛的GFP强度范围内，GFP信号是强敲低（约90%）的可靠报告物（93%-98%）。GFP报告基因使得应用荧光激活细胞分选（FACS）来纯化敲低细胞群体成为可能。这样的群体便于进行蛋白质印迹分析以确定靶蛋白的缺失情况。pG-SUPER还被用于评估通过引入沉默突变而对siRNA产生抗性的外源基因进行的基因替代。核纤层蛋白A的恢复与拯救基因的表达水平呈线性相关。pG-SUPER将通过对敲低和拯救细胞进行简便可靠的检测来扩展基于质粒的siRNA应用。
