Core B

最适合研究的方法是核心B的中心租户和这个项目 MH在表达常见的人类MH突变的小鼠模型中存在。鼠标模型可以提供 多学科团队进行分子、生化、细胞和生理分析所需的足够组织 他们的共同目标是了解这些肌病的病因。鼠标的使用也允许这些 分析将在不同的遗传背景下进行。 核心B将执行支持所有四个项目所需的重复性任务。这个核心将 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。 他们将把这些构建物导入ES细胞，并在项目确定同源靶向克隆后， 扩增这些克隆用于囊胚注射，并将这些靶向ES细胞注射到囊胚中产生 嵌合体。在项目1和项目3确认生殖系传递后，核心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中提取cDNA构建物，并将它们打包到HSV1(RyRs)或 慢病毒(DHPR)病毒粒子将用于表达突变蛋白和细胞内报告 肌管培养，并根据需要分发给所有4个项目。核心B将使所有的成肌细胞系 杂合子和纯合子MH“敲入”小鼠，并保持发音障碍、异常和异常/异常 细胞系将用于所有4个项目。 核心B将从核心C接收人类MHS肌肉样本，并从 这些样本将用于项目1和4。 除了维持MH“敲入”动物品系外，核心B还将负责杂交 这些动物与C57BL6和BalbC小鼠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将为恶性热疗的研究提供一种真正的综合方法。

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.