Improved silencing vector co-expressing GFP and small hairpin RNA
Improved silencing vector co-expressing GFP and small hairpin RNA
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DOI：
10.2144/04361st02
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发表时间：
2004-01-01
期刊：
影响因子：
2.7
通讯作者：
Borisy, GG
中科院分区：
文献类型：
--
作者：
Kojima, SI;Vignjevic, D;Borisy, GG
小干扰RNA（siRNA）是特异性沉默基因表达的有力工具。我们开发了一种改进的载体pG-SUPER，其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。作为测试系统，我们分析了HeLa细胞中的核纤层蛋白A/C敲低。GFP信号是在宽范围的GFP强度上的强敲低（约90%）的可靠报告物（93%-98%）。GFP报告基因使得荧光激活细胞分选（FACS）的应用成为可能，以纯化敲低的细胞群。这样的群体促进了Western印迹分析以确定靶蛋白的消耗。pG-SUPER还用于评价通过引入沉默突变而对siRNA无效的外源基因的基因置换。核纤层蛋白A的回收率与拯救基因的表达水平线性相关。pG-SUPER将通过简单可靠地检测敲低和拯救的细胞来扩展基于质粒的siRNA应用。
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.