Figure 5

GluN2 and GFP transgene expression in vitro mediated by adeno-associated virus. (A) In this experiment, 293FT cells were transduced with AAV viruses designed to express GFP from a CMV promoter, Flag-GluN2A from the CMV, TRE3G, EFS, or RSV promoters, and Flag-GluN2B and GluN2A/BΔC from a CMV promoter. TRE3G promoter containing viruses were transduced into cells that were also transfected with the pTet-Off plasmid. Forty eight hours after viral transduction, 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 or Flag-GluN2 (Texas Red) transgene expression. Non-transduced cells and cells transfected with pRK5-Flag-GluN2 plasmids were processed as negative and positive ICC controls respectively. AAV designed to express Flag-GluN2 from the CMV, TRE3G, EFS, or RSV promoters did not confer convincing transgene expression. However AAV designed to express GFP or GluN2A/BΔC exhibited expression in > 90% of cells. (Scale bar = 20 μm) (B) Depicts similar images as depicted in A, but at lower magnification. (Scale bar = 50 μm).