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Complementation Tests

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A complementation test is a simple cross to identify whether the two mutations are located on the same gene or different genes. It was first performed by Edward Lewis in the 1940s while working on fruit flies. He developed the test to identify the location and arrangement of different mutations on chromosomes.
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Functional Complementation Analysis (FCA): A Laboratory Exercise Designed and Implemented to Supplement the Teaching of Biochemical Pathways

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Published on: June 24, 2016

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.

Investigative Ophthalmology & Visual Science

|June 20, 2013

Summary

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.

Keywords:

RPE65/Rpe65 mutation visual cycle

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Last Updated: May 10, 2026

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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.