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. 2015 Sep 29;24(23):6811–6825. doi: 10.1093/hmg/ddv386

Figure 5.

Figure 5.

Subretinal delivery of improved dual AAV vectors results in ABCA4 expression in mouse photoreceptors and significant reduction of lipofuscin accumulation in the Abca4−/− mouse retina. (A) Representative Western blot analysis of C57BL/6 retinas (whole retinal lysates) either injected with dual AAV2/8 hybrid ABCA4 vectors (5′ + 3′) or with negative controls (neg). The arrow indicates full-length proteins, the molecular weight ladder is depicted on the left. α-3×flag: Western blot with anti-3×flag antibodies; α-Dysferlin: Western blot with anti-Dysferlin antibodies, used as loading control. (B and C) Representative pictures (B) and quantification (C) of lipofuscin autofluorescence (red signal) in the retinas (RPE or RPE + OS) of either pigmented Abca4+/− mice not injected or injected with AAV as control (Abca4+/−) or pigmented Abca4−/− mice either not injected (Abca4−/−) or injected with dual AAV hybrid ABCA4 vectors (Abca4−/− AAV5′+3′). (B) The scale bar (75 μm) is depicted in the picture. RPE: retinal pigment epithelium; ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer. The arrows indicate lipofuscin signal. (C) Mean lipofuscin autofluorescence in the temporal side of three sections for each sample. Mean autofluorescence in each section was normalized for the length of the underlying RPE. The mean value is depicted above the corresponding bar. Values are represented as mean ± s.e.m. ***p ANOVA < 0.0001. n = 4 eyes for each group. (D) Mean number of RPE lipofuscin granules counted in at least 40 fields (25 µm2)/retina of albino Abca4+/+ mice either not injected (Abca4+/+ not inj) or injected with PBS (Abca4+/+ PBS), and albino Abca4−/− mice injected with either PBS (Abca4−/− PBS) or dual AAV hybrid ABCA4 vectors (Abca4−/− AAV5′+3′). The mean value is depicted above the corresponding bar. Values are represented as mean ± s.e.m. *pANOVA ≤ 0.05; **pANOVA ≤0.01. n = 4 eyes from Abca4+/+ not inj; n = 4 eyes from Abca4+/+ PBS; n = 3 eyes from Abca4−/− PBS; n = 3 eyes from Abca4−/− AAV5′ + 3′. More details on the statistical analysis including specific statistical values can be found in the Statistical analysis paragraph of the Materials and Methods section.