Core B

核心B

• 批准号: 7075000
• 负责人: Paul D Allen
• 金额: $ 21.12万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-14 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7075000
• 关键词: Lentivirus; cell line; culture; gene mutation; gene targeting; genetics; human; intracellular; model; muscle; myoblasts; myotubes; proteins; tissue mosaicism; tissues; virion

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Core B的核心租户，该计划项目是最合适的研究方法 MH处于表达经常看到的人类MH突变的鼠模型中。鼠标模型可以提供 通过多学科团队进行分子，生化，细胞和生理分析的足够组织 其集体目标是了解这些肌病的病因。鼠标的使用也允许这些 分析要在不同的遗传背景中进行。 核心B将执行支持所有四个项目所必需的重复任务。这个核心将 提供一个重要而综合的设施，以从项目1和项目3中接收靶向基因靶向结构。 他们将将这些构建体转染到ES细胞中，并且在项目确定同源靶向克隆之后， 扩增这些克隆进行胚泡注射，并将这些靶向ES细胞注入胚泡中以产生 嵌合体。通过项目1和3确认种系传输后，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中采用cDNA结构，并将其包装到HSV1（RYRS）或 慢病毒（DHPRS）病毒体用于表达突变的蛋白质和细胞内报告基因 Myotube文化并根据需要将其分配给所有4个项目。核心B将从所有人中形成肌细胞细胞系 杂合和纯合MH“敲入”小鼠，并保持剂量障碍，失调和dmedic/失调 所有4个项目都将使用的细胞系。 核心B将从Core C接收人类MHS肌肉样本，并从 这些样本由项目1和4使用。 除了维持MH“敲入”动物线外，核心B还将负责杂交 这些具有C57BL6和BALBC小鼠的动物产生具有不同遗传背景的MH动物 通过此，与所有四个项目完全相同的研究模型的统一和一致的供应 核心将允许真正综合的方法来研究恶性高温。