Mouse models for mitochondrial disorders caused by mutations in mtDNA

mtDNA 突变引起的线粒体疾病小鼠模型

基本信息

批准号：
9324583
负责人：
Mikhail F Alexeyev
金额：
$ 7.5万
依托单位：
UNIVERSITY OF SOUTH ALABAMA
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-06-01 至 2017-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9324583
关键词：
Affect Alabama Animal Model Animals Automation Award Bacterial Artificial Chromosomes Bathing CRISPR/Cas technology Carbon Dioxide Cell Line Cells Chest Code Collection Communities Computer software Computers Consumption Core Facility Cryopreservation Cryopreserved Cell Custom Dimensions Disease model Documentation Endothelial Cells Engineering Equilibrium Equipment Excision Floor Freezing Funding Gel Gene Delivery Generations Genes Genetic Goals Homologous Gene Human Incubators Industry Laboratories Libraries Liquid substance Location Lysine-Specific tRNA Mammalian Cell Medical Medicine Methods Microinjections Mitochondria Mitochondrial DNA Mitochondrial Diseases Modeling Modification Morbidity - disease rate Mouse Cell Line Mus Mutagenesis Mutation Neck Nitrogen Oocytes Pathogenicity Patients Pediatric Hospitals Perception Philadelphia Process Protocols documentation Rattus Reader Reading Recovery Research Research Activity Research Infrastructure Research Personnel Sampling Science Side Source Sterility Subfamily lentivirinae Supportive care System Techniques Temperature Time Transfer RNA Transgenic Animals Tube United States National Institutes of Health Universities Vial device Water animal facility base college cryogenics design disease-causing mutation effective therapy embryonic stem cell equipment acquisition gel electrophoresis human disease induced pluripotent stem cell instrument interest knockout gene mitochondrial DNA mutation mortality mouse model mutant mutation screening parent grant prevent screening tool vapor

项目摘要

Mitochondrial diseases caused by mutations in mitochondrial DNA (mtDNA) are a significant source of morbidity and mortality, yet only supportive care is available to affected patients. This lack of effective therapies can be partly attributed to the lack of faithful animal models for mitochondrial diseases caused by mutations in mtDNA. Two critical obstacles to the availability of mouse models of these diseases are 1) the lack of mouse mtDNA mutations homologous to those found in human disease, and 2) the lack of a method to efficiently generate such mutations. Therefore, the long-term GOALS of the proposed studies are 1) to overcome these obstacles in order to enable routine modeling of human diseases caused by mtDNA mutations, and 2) to provide mouse models of mitochondrial disease to the research community. In this application, we propose to leverage expertise developed at the University of South Alabama (mutagenesis of mouse mtDNA) and the Children’s Hospital of Philadelphia (transmitochondrial mouse models) towards generating models of human diseases caused by mutations in mitochondrially-encoded tRNA genes. Using the tools and techniques developed in the previous funding cycle and in preliminary studies, we will 1) generate an arrayed library of mouse clones carrying mutations in mtDNA, 2) use it to perform a targeted screening for mutations in tRNALeuUUR and in tRNALys, and 3) use these mutations as well as tRNA mutations isolated by us previously for the generation and characterization of transmitochondrial animals. It is anticipated that, if successful, the proposed studies will deliver a powerful impetus to mitochondrial research in general and to research on mitochondrial disease in particular by providing the mitochondrial research community with (1) mouse model(s) for mitochondrial disease caused by mutations in tRNA genes, (2) an arrayed library of mouse clones suitable for targeted screening for mtDNA mutations of interest as well as tools and protocols for generating and screening such libraries, and (3) cell line(s) carrying mouse homolog(s) of human pathogenic mtDNA mutations. In this competitive revision, we request funds to procure equipment that will dramatically facilitate the generation and storage of an arrayed library of mouse cell lines containing mutations in mtDNA. While the generation of a collection of mutant mammalian cell lines is not without precedent, the generation of an arrayed library of cell lines with mutations in mtDNA has not been attempted before. In the previous funding cycle, our attempts to generate and screen a large representative library of mouse mtDNA mutants were hampered by low throughput of cryopreservation and sequencing protocols. The equipment that will be procured with the requested funds will introduce semi-automation into our attempts at cryopreservation of the clones, and will enable storage of clones in an 8x12 array format, which is amenable to medium- and high- throughput manipulation and screening.

线粒体DNA（mtDNA）引起的线粒体疾病是重要的来源发病率和死亡率，但仅适用于受影响的患者。这种缺乏有效疗法可以部分归因于缺乏忠实的动物模型，用于线粒体疾病 mtDNA突变。这些疾病小鼠模型可用性的两个关键障碍是1）缺乏与人类疾病同源的小鼠mtDNA突变，以及2）缺乏一种方法有效产生这种突变。因此，拟议研究的长期目标是1）克服这些障碍，以实现由mtDNA引起的人类疾病的常规建模突变，以及2）为研究界提供线粒体疾病的小鼠模型。在这个应用，我们建议利用南阿拉巴马大学开发的专业知识（诱变小鼠mtDNA）和费城儿童医院（多蒙蒙软骨模型）朝向由线粒体编码的TRNA基因突变引起的人类疾病模型。使用在上一个融资周期和初步研究中开发的工具和技术，我们将1）在mtDNA中生成一个载有突变的鼠标克隆的库，2）使用它执行目标筛选trnaleuur和trnalys中的突变，3）使用这些突变以及tRNA突变以前是我们分离出来的，用于产生和表征多蒙膜软骨动物。预计如果成功的话，拟议的研究将为线粒体研究带来强大的动力并通过为线粒体研究界提供有关线粒体疾病的研究（1）由tRNA基因突变引起的线粒体疾病的小鼠模型，（2）小鼠克隆适用于目标筛查的目标筛选以及感兴趣的MTDNA突变以及工具和协议生成和筛选此类文库，以及（3）携带人类致病性小鼠同源物的细胞系（S） mtDNA突变。在此竞争性修订中，我们要求资金采购将极大地促进该设备的设备在mtDNA中包含突变的小鼠细胞系的阵列库的生成和存储。而生成突变哺乳动物细胞系的集合并非没有先例，以前尚未尝试过具有mtDNA突变的细胞系库。在以前的资金中循环，我们试图生成和筛选大型小鼠mtDNA突变体的代表性库是由冷冻保存和测序方案的低吞吐量阻碍。将是与请求的资金一起购买的将在我们对冷冻保存的尝试中引入半自动化克隆，并将以8x12阵列格式存储克隆，该克隆适合中等和高吞吐量操作和筛选。