Protein expression of PRPH2 mutants linked to adRP.

<p>(A-F) Immunohistology of retinas transduced with PRPH2 minigenes containing single point mutations as indicated under the control of the hRHO promoter. Scale bar represents 20 μm. (G) Western blot analysis from membrane preparations of four pooled murine retinas from four animals transduced with the PRPH2 minigenes shown in (A-F) on P14. All retinas were collected three weeks post injection. The arrowhead indicates a degradation band detected at 42 kDa. Ctrl, protein lysates from non-injected control retinas. (H) Semi-quantitative analysis of the results shown in (G). For quantification, three technical replicates were conducted and PRPH2 expression was normalized to the ATPase expression. All data are given as mean values and error bars represent the SEM. Statistical analysis was performed using one-way ANOVA followed by the Dunett’s test. *, p< 0.05; **, p< 0.01; ***, p< 0.001. n.s., not significant.</p

Impaired targeting of truncated PRPH2.

<p>Immunohistology of transduced murine retinas showing rod- (B) and cone-specific (C) expression of a truncated version of PRPH2. (A) Truncated PRPH2 contains only exon 1 and a downstream stop codon (indicated by “X”) mimicking translation from unspliced PRPH2 mRNA and PRPH2 mRNA with intron 1 retention. Staining for B1a and M-ops was used to label rod and cone photoreceptors, respectively. Truncated PRPH2 is not transported to outer segments and is almost exclusively present in inner segments and somata of photoreceptors. Scale bar represents 20 μm.</p

Splice analysis of PRPH2 WT and mutant minigenes in rods and cones.

<p>(A) Representative RT-PCR from cDNA generated from total RNA three weeks post injection from retinas injected with wild-type and mutant rP-mg (left) or cP-mg (right) on P14. Ctrl, control containing the cDNA from non-transduced retina. The single bands of the relevant splice products are numbered (1–4) and highlighted by arrowheads. (B) Schematic representation of the detected splice variants using primers binding to the 3’-end of citrine and to the 5’-end of exon 3 as indicated by the arrows. The numbers of the constructs correspond to the bands marked in (A). (C-E) Semi-quantitative analysis of the relative intensities of the unspliced (C), intron 1 retention (D), and correctly spliced (E) PRPH2 transcripts. For each PRPH2 minigene, the mean percentage of the intensities of these three variants relative to the total intensity (given as sum of the single intensities) was calculated from five RT-PCR analyses conducted with a variable number of cycles (25–27 for rods and 30–32 for cones, respectively). Significance test of the rod or cone mutants to the corresponding wild type (one-way ANOVA followed by Dunett’s test) was performed for rP-mg and cP-mg, respectively. All data were shown as mean values and the error bars represent the standard error of the mean (SEM). *, p< 0.05; **, p< 0.01; ***, p< 0.001. DS, splice donor site.</p