Genetic alteration of the mouse mitochondrial genome and effects on gene expression

The mitochondrial DNA (mtDNA) is a 16kb circular DNA molecule encoding rRNAs, tRNAs and 13 proteins involved in oxidative phosphorylation (OXPHOS). Mutations in these genes can cause a variety of maternally inherited disorders involving neuromuscular and endocrine systems. The detailed analysis of the pathophysiology of these conditions is limited without available means to introduce altered mtDNA into an animal model system. These studies report the transfer of two different exogenous mitochondrial genomes through the germline of the mouse and the production of the first mouse model for human mitochondrial disease. A female embryonic stem (ES) cell line was treated with a lethal dose of the lipophilic dye, rhodamine-6-G (R-6G). R-6G irreversibly disrupts the function of the organelle, rendering the cells auxotrophic for both pyruvate and uridine. The R-6G treated ES cells were rescued by electrofusion with cytoplasts prepared from enucleated fibroblast cells containing either New Zealand Black (NZB) or chloramphenicol-resistant (CAPR) mtDNAs. ES cells predominantly homoplasmic for either of the introduced genomes were injected into blastocyts to generate chimeric animals. The CAP R mtDNAs have a single mtDNA mutation in the 16s rRNA gene that causes a decrease in mitochondrial protein translation and a resulting defect in OXPHOS. The NZB mtDNA has 108 polymorphic differences compared to the 129/Sv mtDNA. An ophthalmological phenotype was discovered in high percentage chimeric mice derived from the CAPR ES cells. The resulting progeny exhibit severe growth retardation and death before 12 days of age. The mice derived from ES cells with NZB mtDNA develop normally with no overt phenotype. To analyze the resulting mice and the cell lines used to develop them, a mitochondrial-specific DNA microarray was developed that contains 450 genes related to mitochondrial function and biogenesis. Analysis of mouse tissues and cell lines has revealed different patterns of mitochondrial gene expression depending on the mtDNA genotype of the sample and, in the case of cell lines, growth conditions.