Figure 2.
Conditional KO of Klf4 gene in the retina using rAAV2-Cre-GFP. A, Schematic of the AAV2-Cre-EGFP vector. L-ITR: Left inverted terminal repeat; R-ITR: right inverted terminal repeat; Cre: Cre recombinase; ires: internal ribosome entry site; EGFP: enhanced green fluorescent protein; hGH poly A: bGH Poly adenylation signal. B, C, Immunostaining of ON longitudinal sections of one month after AAV-Cre-ires-EGFP injection mice showed DAPI-labeled ON resident cells (B) and GFP-labeled axon bundles (C). D–G, Immunostaining of retinal whole mounts at one month after AAV-Cre-ires-EGFP injection showed DAPI-labeled cell nuclei (D), GFP-positive cells (E), Thy1.2-positive RGCs (F), and co-localization of GFP in RGCs shown in merged image (G). H–K, Corresponding longitudinal sections showing DAPI-positive nuclei in all three retinal layers (H), GFP-expressing cells in RGC layers (I), stained positive for Thy1.2 RGC marker (J), and all three channels merged (K); n = 3 eyes. L, Immunoblotting of retina samples obtained one month after intravitreal injection of scAAV-CMV-Cre-ires-EGFP into Klf4fl/fl mice shows selective expression of GFP and decreased expression of KLF4 compared with control retina. The housekeeping GAPDH expression is shown in injected versus control retina. M, Box-whisker plot shows ImageJ quantitation of KLF4 knock-down in AAV-cre-GFP versus control. Box-plot elements include, center line, median; box limits, upper 75th and lower 25th percentile of the data; whiskers, lowest and highest data points. Samples derived from the same experiment and that gels/blots were processed in parallel for quantitative analysis. ONL, outer nuclear layer; INL, inner nuclear layer; RGC, RGC layer (***p = 0.0001–0.0009; unpaired Student’s t test; n = 6 eyes/group). Figure Contributions: Venu Talla acquired the data and generated the figure.