Figure 3
From: Novel 199 base pair NEFH promoter drives expression in retinal ganglion cells
EGFP expression following intravitreal (IV) and subretinal (SR) injection of AAV delivered NEFH promoter constructs utilising EGFP immunocytochemistry. 1 × 109 vg of CMV-P, NEFH-P, hA-P, hF-sp-hA-P or hF-hA-P vectors were delivered by IV or SR injection to adult wild type mice and analysed 4 weeks post-injection. For histology, eyes were enucleated, fixed in 4% pfa and cryosectioned. EGFP immunocytochemistry was performed utilising chicken anti-EGFP primary (Abcam), Alexa-Fluor-488 conjugated secondary antibodies (green) and DAPI nuclear stain (blue). Strong label was detected with IV delivery for CMV-P (A), NEFH-P (C) and hA-P (E) and with SR delivery in CMV-P (B) samples; there was no label in the uninjected retinas (K). At higher sensitivity levels [HS in labels; (L–O)], minimal transduction of photoreceptor cells with SR delivery of hA-P (M) and hF-sp-hA-P (N) and retinal ganglion cell with IV delivery of hF-hA-P (O) constructs was detected (white arrowheads; white rectangles in F, H and I indicate the areas depicted at HS in (M), (N) and (O), respectively); note the high background in the HS images as demonstrated by the uninjected retina (L). ONL outer nuclear layer, INL inner nuclear layer, GCL ganglion cell layer. Scale bar (J) 50 μm.
