In vitro and in vivo functional assays

点击翻译按钮获取中文摘要

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.