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Updated: Apr 15, 2026
Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing
Published on: February 10, 2023
A method for mutagenesis of mouse mtDNA and a resource of mouse mtDNA mutations for modeling human pathological
Rafik Z Fayzulin1, Michael Perez2, Natalia Kozhukhar2
1Department of Cell Biology and Neuroscience, University of South Alabama, Mobile, AL 36688, USA.
Abstract:
Mutations in human mitochondrial DNA (mtDNA) can cause mitochondrial disease and have been associated with neurodegenerative disorders, cancer, diabetes and aging. Yet our progress toward delineating the precise contributions of mtDNA mutations to these conditions is impeded by the limited availability of faithful transmitochondrial animal models. Here, we report a method for the isolation of mutations in mouse mtDNA and its implementation for the generation of a collection of over 150 cell lines suitable for the production of transmitochondrial mice. This method is based on the limited mutagenesis of mtDNA by proofreading-deficient DNA-polymerase γ followed by segregation of the resulting highly heteroplasmic mtDNA population by means of intracellular cloning. Among generated cell lines, we identify nine which carry mutations affecting the same amino acid or nucleotide positions as in human disease, including a mutation in the ND4 gene responsible for 70% of Leber Hereditary Optic Neuropathies (LHON). Similar to their human counterparts, cybrids carrying the homoplasmic mouse LHON mutation demonstrated reduced respiration, reduced ATP content and elevated production of mitochondrial reactive oxygen species (ROS). The generated resource of mouse mtDNA mutants will be useful both in modeling human mitochondrial disease and in understanding the mechanisms of ROS production mediated by mutations in mtDNA.
Insights
Researchers developed a new method to create mouse models with mitochondrial DNA (mtDNA) mutations, aiding the study of diseases like Leber Hereditary Optic Neuropathy (LHON) and aging.
Area of Science:
- Mitochondrial genetics
- Molecular biology
- Disease modeling
Background:
- Mitochondrial DNA (mtDNA) mutations are linked to various diseases including neurodegeneration, cancer, diabetes, and aging.
- A lack of faithful transmitochondrial animal models hinders understanding of mtDNA mutation contributions to these conditions.
Purpose of the Study:
- To develop a method for isolating mouse mtDNA mutations.
- To generate a resource of cell lines for creating transmitochondrial mice to model human diseases.
Main Methods:
- Limited mutagenesis of mouse mtDNA using proofreading-deficient DNA polymerase gamma.
- Segregation of heteroplasmic mtDNA populations via intracellular cloning.
- Generation of over 150 cell lines, including those with disease-relevant mutations.
Main Results:
- Successfully generated a collection of over 150 mouse mtDNA mutant cell lines.
- Identified nine cell lines with mutations mirroring human disease positions, including an ND4 mutation causing Leber Hereditary Optic Neuropathy (LHON).
- Mouse cybrids with the LHON mutation showed decreased respiration, ATP levels, and increased mitochondrial reactive oxygen species (ROS), similar to human patients.
Conclusions:
- The developed method provides a valuable resource for creating mouse models of mitochondrial diseases.
- These models will facilitate research into the mechanisms of mitochondrial disease and ROS production.
- The study advances the understanding of mtDNA mutation impact on cellular function and disease pathogenesis.
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