Genetic Ablation of DHDDS in Photoreceptors in Mouse

Abstract

Purpose: : We have recently identified the K42E mutation in DHDDS (dehydrodolichol diphosphate synthase) as the cause of retinitis pigmentosa (RP) phenotype in a single family with non-syndromic recessive RP. To further study dehydrodolichol diphosphate synthase in the retina, we genetically ablated DHDDS in mouse.

Methods: : Mouse embryonic stem (ES) cells containing the DHDDS^tm1a allele (clone Dhdds_D05) were obtained from UC Davis KOMP Repository. This allele has a trapping cassette "SA-βgeo-pA" (splice acceptor-beta-geo-polyA) flanked by flippase recombinase (Flp) target FRT sites upstream of exon4, resulting in truncation of the endogenous transcript and thus creating a constitutive null mutation. The cassette also tags the gene with a lacZ reporter. The FRT flanked region can be removed by Flp, but exon4, a critical exon, remains floxed. Exon4 can be further removed by the Cre recombinase to achieve conditional knock-out.

Results: : The ES cells were injected into blastocysts and 5 chimeric mice were born. Crossing the chimeric mice with wild type mice produced two F1 mice carrying the DHDDS^tm1a allele. Intercrossing DHDDS^tm1a/+ mice produced more DHDDS^tm1a/+. However, no homozygous DHDDS^tm1a (DHDDS^-/-) mice were obtained even after extensive crossing. The absence of homozygous DHDDS^tm1a may suggest that DHDDS^-/- is lethal. Morphological analysis of retinas from DHDDS^tm1a/+ mice showed normal development of photoreceptors. Strong β-gal staining was found in the photoreceptors as well as in cells in the inner nuclear layer and retinal; ganglion cells. Functional analysis indicates normal ERGs from DHDDS^tm1a/+ mice. Some DHDDS^tm1a/+ mice were crossed with the Rosa26-FLP1 mice that express Flp under the control of Rosa26 to remove the "SA-βgeo-pA" cassette to create DHDDS^fl/fl mice, which express wild-type DHDDS. The DHDDS^fl/fl are been crossed with mice expressing Cre recombinase under the control of the long-mouse opsin promoter (LMOP-Cre mice) to create DHDDS conditional knock-out mice in which DHDDS is ablated only in photoreceptors.

Conclusions: : We have successfully ablated DHDDS in mouse. DHDD^+/- is phenotypically normal but DHDDS^-/- is lethal. DHDDS^fl/fl mice have been created for conditional knock-out of DHDDS in photoreceptors.

Keywords: photoreceptors • retinal degenerations: cell biology • retinal degenerations: hereditary