Generation of novel, tractable conditional knockout models of RP59

Abstract

Purpose : Dehydrodolichyl diphosphate synthase (DHDDS) catalyzes the committed step for dolichol synthesis, required for protein N-glycosylation. Its mutations underlie RP59 (OMIM# 613861). Here, we describe development of novel RP59 mouse models, using Bestrophin1 (BEST1)- and cone-rod homeobox (CRX) Cre-driven Dhdds ablation.

Methods : Dhdds^flx/flx mice were bred against BEST1-Cre or CRX-Cre mice and against ZsGreen reporter mice to achieve Dhdds ablation in RPE/Müller glia cells or photoreceptor (PR)/bipolar (BP) cell progenitors, respectively. Controls: wildtype (WT) C57BL/6J mice, and CRX-Cre and BEST1-Cre mice bred against ZsGreen reporter mice. Western blot (WB) ±PNGase-F was performed on retinas from PN 4-wk old Dhdds KO vs. WT mice, probing with anti-RHO (1D4). Ectopic Cre expression was monitored using a blue light box. Immunohistochemistry (IHC) against RHO, GFAP, and GLUL, and ERG analysis was performed on all mouse lines (N=3/group). Statistical analysis: Student’s t-test, P<0.05 (significance threshold).

Results : CRX-Cre mice expressed Cre in PR and BP cells. BEST1-Cre-ZsGreen mice showed Cre activity in RPE/Müller cells, with “leaky” expression in BP cells. Whole body green fluorescence revealed ectopic expression. Dhdds ablation using the ectopic BEST1-Cre line led to panretinal degeneration. ERG outcomes were a function of Cre expression in individual lines. BEST1-Cre Dhdds^flx/flx mice exhibited significantly decreased ERG responses, vs. controls.. Remnant retinal cells in leaky Best1-Cre Dhdds^flx/flx mice were mostly ZsGreen-negative. Dhdds deletion in PR/BP progenitor cells caused early-onset retinal degeneration with null ERG responses at PN 4-wk. IHC and TUNEL analysis of PN 4-wk KO retinas revealed >90% loss of PR and BP cells. However, WB (RHO+) results were comparable for mutant vs. WT retinas, indicating no protein N-glycosylation defect.

Conclusions : Use of a ZsGreen reporter background afforded confirmation of targeted vs. ectopic Dhdds, highlighting the importance of including a fluorescent reporter background in studies involving Cre-LoxP system-dependent, spatially restricted ablation of target genes. Retinal degeneration, rather than dysplasia, was observed, despite initiation of early-onset Dhdds ablation. These findings suggest that PR and BP cells are highly sensitive to loss of CPT activity.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.