Mouse model of human diseases caused by mtDNA mutations

描述（由申请人提供）：人类遗传疾病的小鼠模型对于确认特定突变导致疾病，获得这些疾病病理生理学的详细分子理解以及开发和测试潜在疗法至关重要。然而，由于技术上的限制，目前我们还无法建立由线粒体突变引起的人类疾病的精确小鼠模型！DNA。尽管有几个研究小组已经证明，他们可以培育出含有从小鼠组织培养细胞中分离出来的线粒体基因组的小鼠，但目前还没有开发出能够让我们将特定核苷酸变化引入小鼠线粒体基因组的技术。我们建议克服这一技术限制。我们已经在大肠杆菌中稳定地克隆了完整的小鼠线粒体基因组，并表明我们可以在这些克隆体中引入任何想要的突变。我们还证明，我们可以将这些工程线粒体基因组引入缺乏自身mtDNA的酵母细胞的线粒体中，并且这些小鼠mtDNA基因组在这些细胞中被准确地复制。我们现在建议使用这些转基因酵母线粒体作为载体，将工程mtDNA基因组转移回小鼠组织培养细胞的线粒体网络中，然后用于产生具有修改线粒体基因组的小鼠。我们这个项目的具体目标是：1)开发和优化使用转基因酵母线粒体作为线粒体传递载体的方法，以有效地融合到小鼠线粒体网络中；2)确定如何最好地筛选或选择已经用修饰的线粒体基因组转化的小鼠细胞。

DESCRIPTION (provided by applicant): Mouse models of human genetic diseases have been critical for confirming that particular mutations cause disease, for obtaining a detailed molecular understanding of the pathophysiology of these diseases, and for developing and testing potential therapies. Technical limitations, however, currently prevent us from generating accurate mouse models of human diseases caused by mutations in our mitochondria! DNA. Although several groups have shown that they can generate mice that contain mitochondrial genomes isolated from mouse tissue culture cells, the technology has not yet been developed that allows us to introduce specific nucleotide changes into mouse mitochondrial genomes. We are proposing to overcome this technical limitation. We have cloned the complete mouse mitochondrial genome stably in E. coli and have shown that we can introduce essentially any desired mutation into these clones. We have also shown that we can introduce these engineered mitochondrial genomes into the mitochondria of yeast cells devoid of their own mtDNA and that these mouse mtDNA genomes are replicated accurately in these cells. We now propose to use these transgenomic yeast mitochondria as vectors for transferring the engineered mtDNA genomes back into the mitochondrial networks of mouse tissue culture cells, which will then be used to generate mice with modified mitochondrial genomes. Our specific aims for this project are to 1) develop and optimize methods for using transgenomic yeast mitochondria as mitochondrial delivery vectors that will efficiently fuse to mouse mitochondrial networks, and to 2) determine how to best screen or select for mouse cells that have been transformed with modified mitochondrial genomes.