A Novel ‘Knock-In’ Mouse Model for Cone Dystrophy: A Point Mutation in Guca1a Causes a Loss of Cone-Mediated Retinal Function and Photoreceptor Degeneration

Abstract

Purpose: : In order to accurately model cone dystrophy 3 (COD3), caused by an E155G substitution in the guanylate cyclase activating protein 1 (GCAP1), we created knock-in mice that have the same amino acid substitution. Here we present extensive, long-term characterization of the retinal phenotype.

Methods: : Mutant mice, generated using gene targeting, were genotyped using PCR and sequencing. Visual function was assessed using Ganzfeld electroretinography (ERG), and ocular morphology assessed using paraffin and resin-embedded sections. Immunohistochemistry was carried out on cryosections and retinal flatmounts.

Results: : We saw a progressive loss of cone-mediated ERG in heterozygous and homozygous mutant mice; b-wave amplitudes from 12 month-old mice were down to 17 % of wild-type. Mutant mice also lacked a flicker response to 10- and 15-Hz light stimuli, whereas wild-type litter-mates had a normal flicker. Rod-mediated b-wave amplitudes were also reduced in mutant mice, although in 12 month-old homozygous mice the rod dominated ERG was still 44 % of wild-type suggesting the mutation primarily affects cone function. Histological analysis showed significant loss of photoreceptor cells at five months of age, up to 47 % loss in homozygotes. We have also studied the effects of the mutation on rod recovery kinetics, photopigment expression, and retinal morphology.

Conclusions: : The analysis performed to date indicates that mice harbouring an E155G mutation in GCAP1 have a phenotype that is consistent with altered cone cell function. Further evaluation of this model using single-cell suction electrode recording, calcium imaging and biochemical analyses is now in progress.

Keywords: retinal degenerations: cell biology • transgenics/knock-outs • retina: distal (photoreceptors, horizontal cells, bipolar cells)