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. 2017 Dec 5;26(2):524–541. doi: 10.1016/j.ymthe.2017.11.019

Figure 5.

Figure 5

Triple AAV Vectors Drive Full-Length Protein Expression in the Mouse Retina

(A) Fluorescence analysis of retinal cryosections from C57BL/6J mice 2 months following subretinal injection of triple ED-AAV2/8 vectors under the control of the ubiquitous CMV promoter. The pictures with insets at higher magnification are representative of n = 5 injected eyes. Full arrows indicate EGFP and DsRed double-positive PRs. Dashed arrows indicate EGFP-only positive PRs. The scale bar (20 μm) is depicted in the figure. DsRed, Discosoma red fluorescent protein; Merge, overlay of EGFP, DsRed, and DAPI images; RPE, retinal pigmented epithelium; ONL, outer nuclear layer. (B) Western blot (WB) analysis of truncated products in eyecup lysates from C57BL/6J mice 2 months following subretinal injection of the combinations of ED-AAVs. The arrows indicate the following products: #A, ED full-length protein; #C, from AAV 1 + 3. (C) WB analysis of lysates from C57BL/6J eyecup 2 months following subretinal injection of triple AAV2/8 vectors encoding for ED under the control of the ubiquitous CMV, the RPE-specific VMD2, and the PR-specific IRBP promoters. The arrow indicates the full-length protein. (D) WB analysis of eyecup lysates from C57BL/6J mice 2 months following subretinal injection of single and triple AAV2/8 vectors encoding for ED under the control of the ubiquitous promoter CMV. (E) WB analysis of eyecup lysates from C57BL/6J mice 2 months following subretinal injection of single and triple AAV2/8 vectors encoding for ED under the control of the PR-specific promoter IRBP. CMV, cytomegalovirus; VMD2, vitelliform macular dystrophy 2; IRBP, human interphotoreceptors retinoid binding proteins. α-3xflag, WB with anti-3xflag antibodies; Ponceau, staining with Ponceau, used as loading control; α-β-Tubulin, WB with anti-β-Tubulin antibodies, used as loading control. Neg, lysates of eyecups following injection with PBS. The molecular weight ladder is depicted on the left; 100 (D), 150 (B and C), and 200 (E) μg of proteins were loaded.