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Functional Genetic Analysis of the Mammalian Mitochondrial DNA Encoded Peptides: A Mutagenesis Approach

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Functional Genetic Analysis of the Mammalian Mitochondrial DNA Encoded Peptides: A Mutagenesis Approach

María Pilar Bayona-Bafaluy et al. Methods Mol Biol.

Abstract

Animal mitochondria are refractory to transformation. This fact has hampered the study of the oxidative phosphorylation system biogenesis by genetic manipulation of the mitochondrial DNA (mtDNA). In humans, a larger variety of mutants have been obtained from patients with mitochondrial diseases, but still we lack a great portion of the range of potential mutants and there is a major obstacle: Animal models cannot be derived from human mtDNA mutants. Until now the only source of mtDNA mutants in mouse was restricted to some drug-resistant-specific cell lines in which a given mtDNA mutation provided growth advantage in the presence of the inhibitor for a specific complex. To overcome these limitations, the authors have developed a protocol that allows the systematic generation of cells harboring mutations in their mtDNA affecting all types of mitochondrial genes. Chemical mutagenesis followed by mtDNA copy number reduction and the use of large-scale negative selection in duplicate cultures, are the key steps of the strategy used.

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