Morphological analysis of retina from cone and rod conditional IRBP knockout mice

Abstract

Purpose : We previously reported that a deficiency in Interphotoreceptor Retinoid Binding Protein (IRBP) results in slow retinal degeneration and myopia phenotypes in mice. We hypothesized that a conditional knockout of IRBP would be useful for determining the mechanisms of these phenotypes. Here we present the effects of knocking out IRBP in cone and rod photoreceptor cells on retinal morphology of 250 day old mice.

Methods : We attempted to generate IRBP floxed mice via traditional methods using a vendor. The resulting mice had only 1 LoxP site. We inserted the second LoxP site using CRISPR technology at the Emory Transgenic Mouse Facility. The resulting mice were bred to homozygosity and confirmed to have normal function. The IRBP^fl/fl mice were bred to Hrgp-CRE and Rho-iCRE75 lines. The two lines were mated to each other, resulting in mice that are of the following 4 conditional knockout genotypes: non-carriers, cone knockout, rod knockout, and cone+rod knockout. These mice were aged to 250 days old, euthanized, and eyes were collected for morphological examination. H&E sections from the eyes were used for manual counting of outer nuclear layer in 100 um wide boxes at positions inferior and superior to the optic nerve.

Results : Sequence analysis of the CRISPR generated mice showed successful insertion of the second LoxP site. IRBP^fl/fl mice showed normal retinal morphology, function, and IRBP gene expression. ONL layer thickness showed the following: non-carrier IRBP^fl/fl mice had normal thickness, Hrgp-CRE IRBP^fl/fl mice had 96% thickness of non-carriers (P=0.0082), Rho-iCRE75 IRBP^fl/fl mice had 59% thickness of non-carriers (P<0.0001), and Hrgp-CRE Rho-iCRE75 IRBP^fl/fl mice had 39% thickness of non-carriers (P<0.0001).

Conclusions : We have successfully made IRBP floxed mice that work well with CRE mice, that are deficient in IRBP expression in cones+rods, and that have retinal degeneration similar to the IRBP knockout mouse. Mice lacking expression of IRBP in rods show less degeneration than the cone+rod mice, suggesting that cones produce enough IRBP to afford some protection. Mice that lack expression of IRBP from cones have a small but significant loss of ONL thickness compared non-carrier mice.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.