Core B

核心B

• 批准号: 7436120
• 负责人: Paul D Allen
• 金额: $ 8.7万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7436120
• 关键词: Animals; Biochemical; Cell Line; Chimera organism; Complementary DNA; Etiology; Gene Targeting; Genetic; Goals; Herpesvirus 1; Human; Injection of therapeutic agent; Knock-in Mouse; Malignant hyperpyrexia due to anesthesia; Methods; Modeling; Molecular; Mus; Muscle; Muscle Fibers; Mutate; Mutation; Myoblasts; Myopathy; Physiological; Proteins; Reporter; Sampling; Study models; Subfamily lentivirinae; Tissues; Virion; blastocyst; embryonic stem cell; mouse model; programs; transmission process

It is the central tenant of Core B and this program project that the most appropriate method to study MH is in murine models that expresses frequently seen human MH mutations. Mouse models can provide sufficient tissues for molecular, biochemical, cellular, and physiological analyses by a multidiscipiinary team whose collective goal is to understand the etiology of these myopathies. The use of mice also permits these analyses to be performed in diverse genetic backgrounds. Core B will perform the repetitive tasks that are necessary to support all four Projects. This core will provide an important and integrated facility to receive gene-targeting constructs from Project 1 and Project 3. They will transfect these constructs into ES cells and after the projects identify homologously targeted clones, amplify these clones for blastocyst injection and inject these targeted ES cells into blastocysts to produce chimeras. After confirmation of germline transmission by Projects 1 and 3, Core B will produce MH "knock-in" mice to be studied by all four Projects. Core B will take cDNA constructs from Projects 1, 3 and 4 and package them into HSV1 (RyRs) or Lentivirus (DHPRs) virions to be used for expression of mutated proteins and intracellular reporters in myotube cultures and distribute them as needed to all 4 projects. Core B will make myoblast cell lines from all heterozygous and homozygous MH "knock-in" mice and maintain dyspedic, dysgenic and dyspedic/dysgenic cell lines to be used by all 4 projects. Core B will receive human MHS muscle samples from Core C and maintain myoblast cell lines from these samples to be used by projects 1 and 4. In addition to maintaining MH "knock-in" animal lines Core B will take responsibility for interbreeding these animals with C57BL6 and BalbC mice to produce MH animals with different genetic backgrounds The uniform and consistent supply of exactly the same study models to all four Projects by this Core will allow a truly integrated approach to the study of Malignant Hyperthermia.

这是研究核心B和本程序项目的最合适的方法 MH在表达常见的人MH突变的鼠模型中。小鼠模型可以提供 足够的组织供多学科小组进行分子、生物化学、细胞和生理学分析 其共同目标是了解这些肌病的病因。小鼠的使用也允许这些 在不同的遗传背景下进行分析。 核心B将执行支持所有四个项目所必需的重复性任务。核心将 提供一个重要的综合设施，以接收来自项目1和项目3的基因靶向构建体。 他们将把这些构建体导入ES细胞，在项目鉴定出同源靶向克隆后， 扩增这些克隆用于胚泡注射，并将这些靶向ES细胞注射到胚泡中以产生 嵌合体在项目1和3确认生殖系传播后，核心B将产生MH“敲入” 这四个项目都将研究小鼠。 核心B将从项目1、3和4中获得cDNA构建体，并将它们包装到HSV 1（RyR）或 慢病毒（DHPR）病毒体用于在大肠杆菌中表达突变的蛋白质和细胞内报告基因。 肌管培养物，并根据需要将其分发给所有4个项目。核心B将使成肌细胞系从所有 杂合和纯合MH“敲入”小鼠，并维持发育不良、遗传不良和发育不良/遗传不良 所有4个项目都将使用细胞系。 核心B将接受来自核心C的人MHS肌肉样品，并维持来自核心C的成肌细胞系。 这些样品将用于项目1和项目4。 除了维持MH“敲入”动物系外，核心B将负责杂交育种 这些动物与C57 BL 6和BalbC小鼠产生具有不同遗传背景的MH动物 统一和一致地向所有四个项目提供完全相同的研究模型， 核心将允许一个真正的综合方法来研究恶性高热。