A method for mutagenesis of mouse mtDNA and a resource of mouse mtDNA mutations for modeling human pathological conditions.
Bottom Line: 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.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).
Affiliation: Department of Cell Biology and Neuroscience, University of South Alabama, Mobile, AL 36688, USA.Show MeSH
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Mentions: For initial validation of the generated collection of mtDNA mutants, baseline respiration rates were examined in 15 clones containing homoplasmic missense mutations with a Seahorse XF-24 extracellular flux analyzer. As expected of an unbiased collection generated without enrichment for functional mutants, clones exhibited a range of respiratory activities with 60% of the mutants retaining WT rates of oxygen consumption, and the remaining 40% demonstrating various degrees of impairment of respiratory chain activity in vivo (Figure 4A).
Affiliation: Department of Cell Biology and Neuroscience, University of South Alabama, Mobile, AL 36688, USA.