Figure 4

The CMV, TRE3G, EFS and RSV promoters differ in their ability to confer GluN2A expression in vitro . (A) Representative ICC images for Flag-GluN2A expression and associated GFP and DAPI staining from transfected cells. In this experiment, AAV plasmids designed to express GluN2A from one of the following promoters, (CMV, TRE3G, EFS, RSV), were cotransfected into 293FT cells with a plasmid containing a CMV-GFP transgene and the pTet-Off plasmid. Twenty four hours after transfection, native GFP expression was observed via fluorescence microscopy and Flag-GluN2 expression was observed via ICC and fluorescence microscopy. Images depict DAPI stained nuclei with the same fields viewed for GFP and Flag-GluN2 (Texas Red) transgene expression. GFP was used as a transfection efficiency control. (B) Quantitation of Flag-GluN2A expression data presented in (A). The Texas Red (Flag-GluN2A) expression levels were normalized to GFP expression levels and these data were plotted as average percent expression as compared to the control group. n = 6, Error bars represent standard error of the mean (SEM). The AAV-CMV-GluN2A plasmid conferred similar Flag-GluN2A expression as the positive control (pRK5-Flag-GluN2A) and these exhibited higher levels of Flag-GluN2A expression, as compared to TRE3G, EFS, and RSV GluN2A containing plasmids. (C) p values for one-way ANOVA with Fisher’s PLSD post-hoc test for (B). Differences were considered significant if, p < 0.05. (D) Mean OD for GFP expression and DAPI staining for Flag-GluN2A expression data presented in (A). One-way ANOVA revealed that GFP levels (F(4,25) = 1.532; p = 0.2235) and DAPI levels (F(4,25 = 1.652; p = 0.1925), did not differ significantly indicating that transfection efficiency did not differ among the groups and the number of cells quantified did not differ significantly among the groups.