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Complementation test of Rpe65 knockout and tvrm148
Charles B Wright1, Micah A Chrenek, Stephanie L Foster
1Department of Ophthalmology, School of Medicine, Emory University, Atlanta, Georgia 30322, USA.
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(KO/KO) (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(-/-) or Rpe65(-/-) 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.
Insights
The tvrm148 mutation in mice causes retinal degeneration by affecting the RPE65 gene, leading to a complete loss of 11-cis-retinal and severe visual impairment. This study confirms tvrm148 is a null allele of RPE65.
Area of Science:
- Genetics
- Ophthalmology
- Molecular Biology
Background:
- A mouse mutation, tvrm148, was previously linked to retinal degeneration.
- Genetic mapping suggested tvrm148 and Rpe65 are located near each other, but their precise relationship was unclear.
Purpose of the Study:
- To determine if the tvrm148 mutation affects the RPE65 gene or a nearby gene.
- To investigate the impact of the tvrm148 mutation on visual function, retinal morphology, and retinoid levels, specifically 11-cis-retinal.
Main Methods:
- Complementation tests were performed using tvrm148 and RPE65 knockout alleles.
- Retinoid levels were quantified using High-Performance Liquid Chromatography (HPLC).
- Visual function was assessed via optokinetic tracking (OKT) and electroretinography (ERG).
- Retinal morphology was examined using light and transmission electron microscopy.
- Rpe65 gene expression and protein levels were measured using qRT-PCR and immunoblotting.
Main Results:
- The tvrm148 and RPE65 knockout alleles failed to complement, indicating they affect the same gene.
- No 11-cis-retinal was detected in tvrm148/tvrm148 (T(-/-)) or Rpe65(-/-) mice.
- T(-/-) mice exhibited significantly reduced visual acuity and ERG responses compared to controls.
- Significant reduction in outer nuclear layer thickness was observed in T(-/-) mice.
- Rpe65 mRNA levels were normal in T(-/-) mice, but protein levels were drastically reduced, suggesting post-transcriptional regulation or protein instability.
Conclusions:
- The tvrm148 mutation is located within the RPE65 gene.
- tvrm148 represents a null allele of RPE65, causing a severe deficiency in 11-cis-retinal.
- The findings provide comprehensive evidence of the mutation's impact on visual function, morphology, and molecular aspects of the retina.
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