Creating a Mouse Model for Human EORD Caused by the R91W Mutation In RPE65: Initial Results

Abstract

Abstract: : Purpose: R91W is the most common missense mutation in Rpe65 leading to early onset retinal dystrophy. Human and mouse RPE65 share 94% identity and position 91 is in one of the most conserved regions of the protein. To understand the mechanism of the human pathology, a Rpe65_R91W knock–in mouse was generated. Methods: ES cells (129S6/SvEvTac) were transfected with a vector containing the first 6 exons and intervening introns of the mouse Rpe65 gene carrying the R91W mutation, a floxed neo– and a diphtheria toxin cassette. Positive ES clones were injected into C57BL/6 blastocysts. Germ–line transmission in a chimeric male was assessed by breeding with C57BL/6 females. Resulting heterozygous offspring was bred with Rpe65^–/– mice to obtain Rpe65^tmR91W/– and Rpe65^wt/– mice for initial tests, including analysis of: 1) expression of the Rpe65 by RT–PCR, Western blotting and immunohistochemistry 2) retinoid composition by HPLC 3) rhodopsin by spectrophotometry, and 4) morphology by light microscopy. Results: Immunohistochemistry showed that the mutated RPE65 protein was indeed expressed in the RPE. This was confirmed by RT–PCR and Western blotting, yet these methods revealed that both mRNA and protein were reduced in the Rpe65^tmR91W/– mice as compared to Rpe65^wt/– mice. Rhodopsin was detectable in Rpe65^tmR91W/– after overnight dark adaptation, however, at levels reduced by about 90–99% as compared to Rpe65^wt/– mice. Furthermore, HPLC profiling of retinoids showed decreased 11–cis retinal levels and massive accumulation of retinyl esters in dark–adapted Rpe65^tmR91W/– mice. Light microscopy revealed lipid–like inclusions in the RPE and showed disturbed photoreceptor outer segment morphology. Conclusions: These initial results demonstrate that the mutated protein is expressed, even though in reduced amount. Presence of rhodopsin and 11–cis retinal suggests that the mutated protein supports isomerohydrolase activity, which is completely blocked in the absence of RPE65, although with very low efficiency. Pure background breeding of the mutated mouse line is currently performed for a detailed analysis.

Keywords: retinoids/retinoid binding proteins • retinal pigment epithelium • retinal degenerations: hereditary