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Charles B. Wright, Micah A. Chrenek, Jeffrey H. Boatright, John M. Nickerson; The Rd12 Allele Of Rpe65 Has A Weak Negative Effect On Vision. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3344.
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Rpe65 knockout (KO), rd12, and TVRM148 mice are three reported models of recessive, loss-of-function Rpe65 deficiency. Closer phenotypic examination of these mutants reveals rd12 mice experience an earlier visual function loss than other Rpe65 mutants. We sought to determine if the nonsense rd12 mutation slightly negatively affects the visual system and how that might occur.
Optokinetic tracking (OKT) and electroretinography (ERG) were used to assess the visual acuity and retinal function of C57BL/6 (+/+), Rpe65 KO (KO/KO), rd12 (rd12/rd12), rd12 heterozygote (rd12/+), Rpe65 KO heterozygote (KO/+), Rpe65 KO/rd12 compound heterozygote (KO/rd12), TVRM148 heterozygote (T/+), and TVRM148 (T/T) mice. Quantitative measurements on outer nuclear layer (ONL) thickness were performed on retina cross-sections. At P30, western blotting with an N-terminal 1-43 amino acid specific antibody was used for Rpe65 protein detection, and steady-state Rpe65 RNA levels were detected through qRT-PCR. Data were analyzed with analysis of variance (ANOVA) with post-hoc Student Newman-Keuls testing.
+/+, KO/+, and T/+ mice had a constant visual acuity (0.38 c/d, or 100%) through P210. rd12/+ mice had 92% visual acuity at P30 but declined to 79% visual acuity by P210. KO/KO and T/T mice lost 50% visual acuity by P120 and had no detectable visual acuity by P210. Conversely, rd12/rd12 mice lost detectable visual acuity at a much earlier age, with 50% visual acuity at P60 and 0% by P120. KO/rd12 mice lost visual acuity similarly to rd12/rd12 mice (79% at P30 and 0% at P120). ERG results parallel OKT results. Comparison of +/+, KO/KO, rd12/rd12, and T/T retina morphologies showed mutant lines experience ONL thinning but preserved architecture. qRT-PCR showed rd12/rd12 Rpe65 mRNA does not undergo nonsense-mediated decay, but western blotting showed no detectable 43 amino acid peptide.
Visual function loss in rd12/rd12, rd12/+, KO/rd12 mice suggests the rd12 mutation exerts a slight negative effect on the visual system. KO/+ and T/+ OKT and ERG data suggests that the rd12/+ visual function loss is not the result of Rpe65 haploinsufficiency. The presence of the rd12 Rpe65 transcript, not of an Rpe65 protein fragment, may be responsible for a mild gain-of-function toxicity in the visual system.
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