Tropisms of AAV for Subretinal Delivery to the Neonatal Mouse Retina and Its Application for In Vivo Rescue of Developmental Photoreceptor Disorders
Figure 3
Gene expression analysis of the Crx KO retina treated with AAV-Crx.
(A) Schematic diagram of the AAV2/5-Crx2kb-Flag-Crx construct. (B) Expression analysis of Crx by RT-qPCR using RNA isolated from control and AAV-treated Crx KO retinas (Control Crx KO retinas: n = 3 from three different mice and AAV-treated Crx KO retinas: n = 4 from four different mice). (C) Western blot analysis of FLAG-CRX using control and AAV-treated Crx KO retinas from three different mice respectively. An anti-FLAG antibody was used to detect FLAG-CRX. ACTB (β-actin) was used as a loading control. (D–F) Immunostaining of the Crx KO retinas treated with AAV-Crx or PBS using an anti-FLAG antibody. The distribution of FLAG-CRX expression in control and AAV-Crx treated Crx KO retinas (E, F). Enlarged images in white boxes (a-c and d-f) in Figure 3E and F, respectively. Scale bar represents 50 µm (D) and 1 mm (E, F). (G–Q) Expression analyses of eleven genes related to human retinal diseases three weeks after treatment (Control Crx KO retinas: n = 3 from three different mice and AAV-treated Crx KO retinas: n = 4 from four different mice). Rhodopsin (G), Gnat1 (H), S-opsin (I), M-opsin (J), Pde6g (K), Slc24a1 (L), Rdh12 (M), Rpgrip1 (N), Nrl (O), Cabp4 (P), and Fscn2 (Q). Control retinas were injected with PBS (Vehicle). Rpl4 was used for normalization. Primers for qPCR were listed in Table S1. Error bar represents the SD from the means of three control retinas and four treated retinas. **p<0.01, *p<0.05. ITR: inverted terminal repeat, RPE: retinal pigment epithelium, ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion cell layer.