Complementation Test of <em>Rpe65</em> Knockout and Tvrm148

Charles Wright

Micah Chrenek

Stephanie Foster

Todd Duncan

Supplementary Material

Supplemental Data:

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^{Department of Ophthalmology, School of Medicine, Emory University, Atlanta, Georgia}

^{Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland}

^{Rehabilitation Research and Development Center of Excellence, Atlanta VA Medical Center, Decatur, Georgia}

Correspondence: John M. Nickerson, Department of Ophthalmology, Emory University, B5602, 1365B Clifton Road NE, Atlanta, GA 30322; ude.yrome@njtil.

Received 2013 May 1; Accepted 2013 Jun 10.

Abstract

Purpose.

A mouse mutation, tvrm148, was previously reported as resulting in retinal degeneration. Tvrm148 and Rpe65 map between markers D3Mit147 and D3Mit19 on a genetic map, but the physical map places RPE65 outside the markers. We asked if Rpe65 or perhaps another nearby gene is mutated and if the mutant reduced 11-cis-retinal levels. We studied the impact of the tvrm148 mutation on visual function, morphology, and retinoid levels.

Methods.

Normal phase HPLC was used to measure retinoid levels. Rpe65^+/+, tvrm148/+ (T^{), tvrm148/tvrm148 (T}^{), RPE65}⁽Rpe65^−/−), and Rpe65^T/− mice visual function was measured by optokinetic tracking (OKT) and electroretinography (ERG). Morphology was assessed by light microscopy and transmission electron microscopy (TEM). qRT-PCR was used to measure Rpe65 mRNA levels. Immunoblotting measured the size and amount of RPE65 protein.

Results.

The knockout and tvrm148 alleles did not complement. No 11-cis-retinal was detected in T^orRpe65^−/− mice. Visual acuity in Rpe65^+/+ and T^{mouse was ∼0.382 c/d, but 0.037 c/d in T}^{mice at postnatal day 210 (P210). ERG response in T}^{mice was undetectable except at bright flash intensities. Outer nuclear layer (ONL) thickness in T}^{mice was ∼70% of}Rpe65^+/+ by P210. Rpe65 mRNA levels in T^{mice were unchanged, yet 14.5% of}Rpe65^+/+ protein levels was detected. Protein size was unchanged.

Conclusions.

A complementation test revealed the RPE65 knockout and tvrm148 alleles do not complement, proving that the tvrm148 mutation is in Rpe65. Behavioral, physiological, molecular, biochemical, and histological approaches indicate that tvrm148 is a null allele of Rpe65.

Keywords: RPE65/Rpe65, visual cycle, mutation

Abstract

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Acknowledgments

Supported by National Institutes of Health (NIH) Grants P30EY006360, R01EY016470, T32EY007092, R01EY014026, R01EY016435, R01EY021592, and a grant from the Abraham J. & Phyllis Katz Foundation (JHB), an unrestricted Departmental Award from Research to Prevent Blindness, the Intramural Research Program of the National Eye Institute, NIH (Z01EY000444). The authors alone are responsible for the content and writing of the paper.

Disclosure: C.B. Wright, None; M.A. Chrenek, None; S.L. Foster, None; T. Duncan, None; T.M. Redmond, None; M.T. Pardue, None; J.H. Boatright, None; J.M. Nickerson, None

Acknowledgments

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