March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
Genetic Ablation of DHDDS in Photoreceptors in Mouse
Author Affiliations & Notes
  • Ying Li
    Bascom Palmer Eye Institute, University of Miami, Miami, Florida
  • Zhengying Wang
    Bascom Palmer Eye Institute, University of Miami, Miami, Florida
  • Pingping Chen
    Bascom Palmer Eye Institute, University of Miami, Miami, Florida
  • Lauren N. Correa
    Bascom Palmer Eye Institute, University of Miami, Miami, Florida
  • Byron L. Lam
    Bascom Palmer Eye Institute, University of Miami, Miami, Florida
  • Yiwen Li
    Bascom Palmer Eye Institute, University of Miami, Miami, Florida
  • Rong Wen
    Bascom Palmer Eye Institute, University of Miami, Miami, Florida
  • Footnotes
    Commercial Relationships  Ying Li, None; Zhengying Wang, None; Pingping Chen, None; Lauren N. Correa, None; Byron L. Lam, None; Yiwen Li, None; Rong Wen, None
  • Footnotes
    Support  NIH Grant R01EY018586, P30EY14801; Hope for Vision, the James and Esther King Biomedical Research Program of the State of Florida JEK 08-KN-09; the Department of Defense Grant W81XWH-09-1-0674; RPB.
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 2582. doi:https://doi.org/
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      Ying Li, Zhengying Wang, Pingping Chen, Lauren N. Correa, Byron L. Lam, Yiwen Li, Rong Wen; Genetic Ablation of DHDDS in Photoreceptors in Mouse. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2582. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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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 DHDDStm1a 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 DHDDStm1a allele. Intercrossing DHDDStm1a/+ mice produced more DHDDStm1a/+. However, no homozygous DHDDStm1a (DHDDS-/-) mice were obtained even after extensive crossing. The absence of homozygous DHDDStm1a may suggest that DHDDS-/- is lethal. Morphological analysis of retinas from DHDDStm1a/+ 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 DHDDStm1a/+ mice. Some DHDDStm1a/+ 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 DHDDSfl/fl mice, which express wild-type DHDDS. The DHDDSfl/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. DHDDSfl/fl mice have been created for conditional knock-out of DHDDS in photoreceptors.

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