Figure 2
Intravitreal AAV2-Cre/GFP injection achieved conditional knockout of the Hdac3 gene. (A-B) TEXAS RED labeling of HDAC2 in Rosa26-LacZfl/fl and Hdac3fl/fl AAV2-Cre/GFP injected eyes at 5 days post ONC illustrates nuclear localization of this HDAC in cells of the GCL of both retinas. (C-D) TEXAS RED labeling of HDAC3 in Rosa26-LacZfl/fl and Hdac3fl/fl AAV2-Cre/GFP injected eyes at 5 days post ONC demonstrates the lack of HDAC3 in the Hdac3fl/fl RGCs (Scale bar: 15 μm). (E) QPCR was used to determine abundance of Hdac3 mRNA from pooled retinas of 5 Rosa26-LacZfl/fl and Hdac3fl/fl mice at 4 weeks following AAV2-Cre/GFP injection. In uncrushed retinas, the abundance of Hdac3 mRNA was significantly lower in Hdac3 cKO retinas compared to Rosa26-LacZfl/fl retinas (*P ≤ 0.05). (F) Protein from individual retinas of AAV2-Cre/GFP injected (4-week incubation) and uninjected Hdac3fl/fl mice showed an overall decrease in protein levels in Hdac3 cKO mice, although there was no significant difference in mean abundance (P > 0.05). (G) Pooled retinal protein from 5 AAV2-Cre/GFP injected Rosa26-LacZfl/fl and Hdac3fl/fl mice at 1 and 5 days post ONC were run on a Western blot. ONC results in an increase in HDAC3 accumulation in Rosa26-LacZfl/fl mice by 5 days, but this response is significantly abrogated in Hdac3fl/fl animals (*P ≤ 0.05) when comparing Rosa26-LacZfl/fl and Hdac3fl/fl mice at 5 days.