Browsing by Subject "Restriction endonuclease"

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    Engineering mammalian mitochondrial genomes
    (2012-01) Yang, Yi-Wei
    Mutations in mitochondrial genomes can cause dysfunction of mitochondria and disease. Studying pathogenic mutations in mitochondrial genomes is difficult due to the lack of suitable tools for engineering mitochondrial genomes in mammalian cells. This project aims to solve some of the technical hurdles that will allow direct manipulation of mammalian mitochondrial genomes and characterization of the biological consequences of specific sequence changes in mammalian mitochondrial genomes in vivo. Mouse mitochondrial genomes were cloned and stably maintained in E. coli at low copy number. Using standard techniques of molecular biology, one full and three deleted mouse mitochondrial genome clones carrying a selection marker, γ-ori and yeast COX2 gene flanked by duplicated sequences were generated. These mouse mitochondrial genome clones were stably maintained as circular monomers being transformed into yeast mitochondria. The exogenous sequences used for cloning and screening were removed by homologous recombination in yeast mitochondria. Yeast Artificial Mitochondria (YAM) strains were generated that are yeast cells carrying only engineered mouse mitochondrial genomes in their mitochondria. In order to evaluate the biological consequences of engineered mouse mitochondrial genomes in mouse cells, an artificial cytoplast fusion method was developed for introducing isolated mitochondria into mouse tissue culture cells. This is the first method that can deliver large quantities of isolated mitochondria into mammalian tissue culture cells. The respiratory deficiency phenotype in the recipient cells was rescued by the introduced mouse mitochondria, indicating the procedure can preserve the biological activities of isolated mitochondria. Isolated YAM were introduced into mouse tissue culture cells by the artificial cytoplast cell fusion method. Interestingly, as engineered mouse mitochondrial genomes were actively replicated in yeast, these genomes did not replicate efficiently enough to be maintained in mouse cells in long-term culture. A competition between the rat mitochondrial genomes co-introduced from the artificial cytoplasts (generated from rat oocytes) and the mouse mitochondrial genomes carried in YAM could be the cause. In order to test the hypothesis that the mouse mitochondrial genomes could replicated more efficiently if the mitochondrial genomes from rat oocytes were eliminated from the same mouse cells, artificial heteroplasmic mouse cell lines were generated. By expressing mitochondrial-targeted XhoI endonuclease, the mitochondrial genomes from rat oocytes that contained a single XhoI site were selectively removed from the mouse cells whereas genomes without an XhoI site continued to replicate. The effect of expressing mitochondrial-targeted XhoI endonuclease on XhoI-negative engineered mouse mitochondrial genomes in mouse cells will be investigated in the future.