Improved silencing vector co-expressing GFP and small hairpin RNA.
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.
Kojima
Shin-ichiro
S
Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, 303 E. Chicago Ave., Chicago, IL 60611, USA. s-kojima@northwestern.edu
Vignjevic
Danijela
D
Borisy
Gary G
GG
D020868:Gene Silencing; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D034904:Lamin Type A / Q000235:genetics; D034882:Lamins / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D014162:Transfection / Q000379:methods*
D034904:Lamin Type A; D034882:Lamins; D008164:Luminescent Proteins; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C440308:lamin C; D049452:Green Fluorescent Proteins