In vitro and in vivo functional assays

核心B提供了用于测定BMD样肌营养不良蛋白的生物化学功能的体外和体内测定。这些内部缺失的半功能蛋白质由其他研究项目和核心产生，包括对来自具有良好表征的框内突变的患者的人类细胞中的内源性蛋白质的研究（项目1，3），在vh/o中递送至细胞或小鼠的特定缺失的鼠构建体（项目1），以及通过外显子跳跃疗法实现的框内缺失（项目2）。核心B的具体目标包括由儿童国家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测定。Core B提供的一种创新试验是我们最近发表的使用近红外组织蛋白酶B笼状底物评估肌营养不良活性的活体动物成像方法（Baudy et al 2010）。另一种创新的检测方法是使用MS/MS分析定量分泌组测量膜不稳定性。 资源共享将包括发布和公众访问每项测定的标准操作规程，正如我们与欧盟Treat-NMD网络合作进行的小鼠临床前终点（http：//www.Treat-nmd.eu/research/preclinical/SOPs/）（Nagaraju 2009; Spurney等人2009）。我们还提供 将体内试验作为药物筛选的核心功能提供给外部实验室，并将体外试验提供给外部实验室进行协作。

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.