Analytical Core Unit

在整个计划中，传递促进或破坏生物过程的基因是 对所有4个项目中概述的目标进行起诉。纯化重组蛋白的生产将 促进确定新的翻译后机制(项目1和4)和蛋白质分析： 蛋白质相互作用(项目2)。活性氧和活性氮物种的测定 (超氧化物、过氧化氢和一氧化氮)是必要的，以关联这些水平的变化 代谢产物对其生物效应的影响(项目1-4)。细胞成像主播\提供了新的视角 蛋白质亚细胞定位和共定位的调节(项目2-4)。这些服务是最好的 鉴于其固有的困难、对专门设备和专业知识的依赖以及拟议的用途，集中化 超过4个项目中的2个。为实现这些目标，我们提出了以下具体目标： 特定目标1-基因传递：该目标的目标是构建、纯化和传递重组 腺病毒和慢病毒表达基因和小的抑制RNA，也合成大量 用于体内组织电穿孔的无内毒素质粒DNA。 特定目标2-分析：该目标的目标是a)合成用于TH直接的重组蛋白 分析翻译后修饰和酶活性b)以提供统一的测量 活性氧和氮物种和c)确定蛋白质的亚细胞定位和鉴定- 细胞内共定位。这些服务将由Fulton博士(核心领导)监督，他 在重组病毒的生产和交付以及反应性分析方面有丰富的经验 氮素物种和布莱克博士(共同领导)，他是表达和纯化的专家 蛋白质，使用EPR技术测量纯化蛋白质、细胞培养和整体中的ROS水平 动物。 相关性(请参阅说明)： 这个核心的总体目标是提供一种集中化的服务，通过 重组病毒和电穿孔，提供了活性氮的标准化测量和 生物体液中的活性氧物种，产生大量的重组蛋白和状态 ART细胞成像技术。

Throughout the Program, the delivery of genes to promote or disrupt biological process is integral to the prosecution of the aims outlined in all of the 4 projects. The production of purified recombinant proteins will facilitate the identification of novel post-translational mechanisms (Projects 1 and 4) and analysis of protein: protein interactions (Project 2). The measurement of reactive oxygen and reactive nitrogen species (superoxide, hydrogen peroxide and nitric oxide) is necessary to correlate changes in the levels of these metabolites to their biological effects (Projects1-4). Cellular imaging VJ\\\ provide new perspectives into the regulation of protein subcellular localization and co-localization (Projects 2-4). These services are best centralized given their inherent difficulty, reliance on specialized equipment and expertise and proposed use by more than 2 of the 4 projects. To accomplish these goals, we propose the following specific aims: Specific Aim 1 - Gene Delivery: The goals of this aim are to construct, purify and deliver recombinant adenovirus and lentivirus expressing genes and small inhibitory RNAs and also to synthesize large quantities of endotoxin-free plasmid DNA for the electroporation of tissues in vivo. Specific Aim 2 - Analysis: The goals of this aim are a) to synthesize recombinant proteins for th direct analysis of post-translational modifications and enzymatic activity b) to provide uniform measurements of reactive oxygen and nitrogen species and c) to determine the subcellular localization of and identify protein- colocalization inside cells. These services will be overseen by Dr. Fulton (Core Leader) who has considerable experience with the production and delivery of recombinant viruses and analysis of reactive nitrogen species and by Dr. Black (co-core leader) who is an expert in the expression and purification of proteins, the use of EPR technology to measure ROS levels in purified protein, cell culture, and whole animals. RELEVANCE (See Instructions): The overall goal of this Core is to provide a centralized service that facilitates the delivery of genes via recombinant virus and electroporation, provides standardized measurements of reactive nitrogen and reactive oxygen species in biological fluids, generates large quantities of recombinant proteins and state of the art cellular imaging.