Generating mtDNA mutant mice to model human mitochondrial diseases

项目摘要 人类线粒体DNA（mtDNA）的突变会导致母系遗传性疾病， 包括肌肉和神经系统在内的身体多个部位。超过250种致病性 在人类线粒体DNA中已经发现了突变，导致至少1种疾病的一系列病理变化。 每5000名受试者中。然而，目前尚不清楚mtDNA突变如何导致不同的 临床表型由于目前还没有治愈线粒体疾病的方法， 携带致病性mtDNA突变的模型对于理解 疾病机制和开发治疗方法。缺乏这样的模式， 成为线粒体医学研究的主要障碍。主要挑战是 产生这些模型源于无法操纵线粒体基因组。 因此，本项目建议建立一个管道，以确定特定的小鼠mtDNA突变 与致病人类mtDNA突变同源，选择和表征小鼠细胞 携带这些突变，并产生和验证相应的突变小鼠模型。 我们管道的关键组成部分包括一个大型mtDNA突变小鼠细胞库 理论上包含所有可能的mtDNA突变，高通量高灵敏度mtDNA 突变检测分析，每天可以筛选数万个样本， 胚胎干细胞系，允许快速生成transmitochondrial小鼠系。 利用这一管道，该项目将产生：1）mtDNA突变小鼠成纤维细胞， 含有与人类致病突变同源的突变的胚胎干细胞系， 和2）含有这种突变的mtDNA突变小鼠模型。这些体外和体内的工具 将非常有助于研究线粒体疾病的分子机制， mtDNA突变在其他与体细胞mtDNA突变相关的疾病中的作用 积累这些新工具的生产也将是至关重要的治疗 线粒体疾病的发展。

Project Summary Mutations in human mitochondrial DNA (mtDNA) cause maternally inherited diseases that affect multiple parts of the body including the muscles and nervous system. More than 250 pathogenic mutations have been identified in human mtDNA, causing a spectrum of pathologies in at least 1 in every 5000 subjects. However, it remains unclear how mtDNA mutations lead to disparate clinical phenotypes. As there are currently no cures for mitochondrial diseases, in vivo mouse models harboring pathogenic mtDNA mutations are of critical importance both to understand disease mechanisms and to develop therapeutic approaches. The lack of such models has become a major hurdle to research on mitochondrial medicine. The primary challenge to generate these models originates from the inability to manipulate the mitochondrial genome. Therefore, this project proposes to build a pipeline to identify specific mouse mtDNA mutations homologous to disease-causing human mtDNA mutations, select and characterize mouse cells harboring these mutations, and generate and validate corresponding mutant mouse models. The key components of our pipeline include a large library of mtDNA mutant mouse cells containing theoretically all possible mtDNA mutations, a high-throughput high-sensitivity mtDNA mutation detection assay that can screen tens of thousands of samples per day, and a mouse embryonic stem cell line that allows quick generation of transmitochondrial mouse lines. Using this pipeline, the project will generate: 1) mtDNA mutant mouse fibroblast and embryonic stem cell lines containing mutations homologous to human pathogenic mutations, and 2) mtDNA mutant mouse models containing such mutations. These in vitro and in vivo tools would be highly useful in studying the molecular mechanisms of mitochondrial diseases and the roles of mtDNA mutations in other diseases that are linked with somatic mtDNA mutation accumulation. The production of these novel tools will also be crucial for therapeutic development of mitochondrial diseases.