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.

它是Core B的核心租户，也是这个项目最合适的学习方法 MH 在小鼠模型中表达常见的人类 MH 突变。鼠标型号可提供 足够的组织供多学科团队进行分子、生化、细胞和生理分析 他们的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需的重复任务。该核心将 提供一个重要的综合设施来接收来自项目 1 和项目 3 的基因靶向构建体。 他们将这些构建体转染到 ES 细胞中，并在项目鉴定出同源目标克隆后， 扩增这些克隆用于囊胚注射，并将这些靶向 ES 细胞注射到囊胚中以产生 嵌合体。项目1和项目3确认种系传播后，Core B将产生MH“敲入” 所有四个项目都将研究小鼠。 核心 B 将从项目 1、3 和 4 中获取 cDNA 构建体，并将其包装到 HSV1 (RyRs) 或 慢病毒 (DHPR) 病毒体用于表达突变蛋白和细胞内报告基因 myotube 培养并根据需要将其分发到所有 4 个项目。 Core B 将产生来自所有细胞的成肌细胞系 杂合子和纯合子 MH“敲入”小鼠并维持 Dyspedic、dysgenic 和 Dyspedic/dysgenic 所有 4 个项目均使用的细胞系。 Core B将从Core C接收人类MHS肌肉样本并维持来自Core C的成肌细胞系 这些样本供项目 1 和 4 使用。 除了维持 MH“敲入”动物品系外，Core B 还将负责杂交 这些动物与 C57BL6 和 BalbC 小鼠一起产生具有不同遗传背景的 MH 动物 统一、一致地向所有四个项目提供完全相同的研究模型 Core 将为恶性高热研究提供真正综合的方法。