In vitro and in vivo functional assays

核心B提供了体外和体内测试，以确定BMD样肌营养不良蛋白的生化功能。这些内部缺失的半功能蛋白是由其他研究项目和核心产生的，包括对具有良好特征的框内突变患者的人类细胞内源性蛋白的研究(项目1、3)、在VH/O中传递到细胞或小鼠的特定缺失的小鼠构建(项目1)以及通过外显子跳过疗法完成的框内缺失(项目2)。核心B的具体目标包括由国家儿童医学中心遗传医学研究中心内的五个合作实验室开发的成熟的检测方法。由Core B提供的一种创新测试是我们最近发表的活体动物成像方法，用于使用近红外组织蛋白酶B笼状底物评估肌营养不良症的活动(Baudy等人，2010)。另一种创新的分析方法是使用MS/MS图谱定量测定膜的不稳定性。 资源共享将包括公布和公开每种分析的标准操作程序，就像我们与欧盟针对小鼠临床前终点(http://www.treat-nmd.eu/research/preclinical/SOPs/)的治疗-国家MD网络合作所做的那样(Nagaraju，2009；SPurney等人)。2009年)。我们还提供 体内试验作为药物筛选的核心职能向外部实验室进行，体外试验以协作为基础向外部实验室进行。

Core B provides in vitro and in vivo assays for determining the biochemical functionality of BMD-like dystrophin proteins. These internally deleted semi-functional proteins are generated by the other research projects and cores, and include studies of endogenous proteins in human cells from patients with wellcharacterized in-frame mutations (Projects 1, 3), murine constructs of specific deletions delivered to cells or mice in vh/o (Project 1), and in-frame deletions accomplished by exon-skipping therapeutics (Project 2). The specific aims of Core B include well-established assays developed by the five collaborating laboratories within the Research Center for Genetic Medicine at Children's National Medical Center. An innovative assay offered by Core B is our recently published live-animal imaging method for assessment of muscular dystrophy activity using a near-infrared cathepsin B caged substrate (Baudy et al 2010). An additional innovative assay is quantitative secretome measures of membrane instability using MS/MS profiling. Resource sharing will include publication and public access of standard operating procedures for each assay, as we have done collaboratively with the EU Treat-NMD network for murine pre-clinical endpoints (http://www.treat-nmd.eu/research/preclinical/SOPs/) (Nagaraju 2009; Spurney et al. 2009). We also offer in vivo assays as a core function for drug screening to external laboratories, and in vitro assays to external laboratories on a collaborative basis.