High density mutagenesis of mtDNA

线粒体DNA高密度诱变

• 批准号: 7500862
• 负责人: Mikhail F Alexeyev
• 金额: $ 18.01万
• 依托单位: UNIVERSITY OF SOUTH ALABAMA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-25 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7500862
• 关键词: Acceleration; Adult; Animal Disease Models; Animal Model; Animal Testing; Animals; Antibiotic Resistance; Biogenesis; Cell Line; Cells; Childhood; Chloramphenicol; Collection; Cultured Cells; DNA; DNA polymerase gamma; Data; Development; Disease; Electron Transport; Epidemiologic Studies; Generations; Genes; Goals; Human; Infection; Methods; Mitochondria; Mitochondrial DNA; Mitochondrial Diseases; Modality; Modeling; Mouse Cell Line; Mus; Mutagenesis; Mutation; Nuclear; Oxygen Consumption; Patients; Ploidies; Point Mutation; Prevalence; Production; Protein Overexpression; Rate; Reagent; Reporting; Safety; Symptoms; Syndrome; Technology; Testing; Therapeutic; Today; Transfection; Vitamins; cofactor; density; disease-causing mutation; endodeoxyribonuclease XmaI; functional restoration; gene therapy; human disease; improved; mitochondrial DNA mutation; mouse model; mutant; novel therapeutics; outcome forecast; polypeptide; tissue culture

DESCRIPTION (provided by applicant): Recently, we have succeeded in the reduction of mutant mtDNA content in cells carrying heteroplasmic T8993G mutation, which causes the NARP and MILS syndromes, heritable fatal mitochondrial disorders. The rates of ATP production and oxygen consumption in treated cells improved, while rates of lactate secretion decresed. This fact indicates the functional restoration of electron transfer chain. Our approach, if used therapeutically, has a potential of becoming the first therapy capable of reversing the symptoms for any mitochondrial disorder. However, the novelty of this approach as therapeutic modality makes it imperative to investigate its safety. Ideally, this would be done in experimental animals. Unfortunately, currently there is no animal model for either T8993G NARP, or (with perhaps one exception) any other human disease caused by the mutation in mtDNA. Recently, there was an important breakthrough: several groups demonstrated their ability to generate transmitochondrial mice carrying mutation, which was induced in tissue culture by selecting cells for resistance to antibiotic chloramphenicol. Therefore, if we had cultured mouse cells carrying mutations synonymous to those found in human diseases, we could generate mouse models for these diseases. However, neither such cells, nor methods to generate such cells exist today. Therefore, the goal of this application is to develop a technology for the generation of a collection of mouse cell lines with various mutations in mtDNA, to generate such a collection, and to do an initial characterization of this collection. We plan to first generate random mutations in mouse mtDNA by overexpressing the error-prone mitochondrial DNA-polymerase gamma and then to force cells with random mtDNA mutations through the bottleneck by first depleting and then repleating mtDNA in these cells. If successful, proposed studies will provide a significant breakthrough as they will not only help us to move forward with our NARP studies, but will also dramatically accelerate the search for effective therapies by making animal models for mtDNA diseases routinely available.

描述（由申请人提供）： 最近，我们成功地降低了携带异质T8993G突变的细胞中突变的mtDNA含量，从而导致NARP和MILS综合征，可遗传的致命线粒体疾病。治疗细胞中ATP产生和氧气消耗的速率得到了改善，而乳酸分泌率却衰落。这个事实表明电子传递链的功能恢复。如果使用治疗方法，我们的方法有可能成为第一种能够逆转任何线粒体疾病症状的疗法。但是，这种方法作为治疗方式的新颖性使得必须研究其安全性。理想情况下，这将在实验动物中进行。不幸的是，目前尚无针对T8993G NARP的动物模型，或者（也许是一个例外）MTDNA突变引起的任何其他人类疾病。最近，有一个重要的突破：几个小组证明了它们产生携带突变的多蒙膜软骨小鼠的能力，这在组织培养中是通过选择细胞来耐药性抗生素氯霉素的能力。因此，如果我们对携带突变的培养小鼠细胞代名词，则可以生成这些疾病的小鼠模型。但是，当今这些细胞和产生此类细胞的方法都不存在。因此，此应用程序的目的是开发一种技术，以生成具有MTDNA中各种突变的小鼠细胞系的集合，以生成这种集合，并对此集合进行初始表征。我们计划首先通过过表达易误误的线粒体DNA-聚合酶γ，然后通过首先耗尽瓶颈而迫使细胞在这些细胞中通过瓶颈迫使细胞在这些细胞中通过瓶颈强迫细胞，从而在小鼠mtDNA中产生随机突变。如果成功，提出的研究将提供重大突破，因为它们不仅可以帮助我们继续进行NARP研究，而且还将通过制造用于MTDNA疾病的动物模型来大大加速寻找有效疗法的搜索。