Analytical Core Unit

在整个计划中，传递基因以促进或破坏生物过程是不可或缺的， 实现所有4个项目中概述的目标。纯化的重组蛋白的生产将 促进新的翻译后机制的鉴定（项目1和4）和蛋白质的分析： 蛋白质相互作用（项目2）。活性氧和活性氮的测定 （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， 代谢物的生物学效应（项目1 -4）。细胞成像VJ\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、对专门设备和专门知识的依赖以及拟议的用途， 4个项目中的2个以上。为实现这些目标，我们提出以下具体目标： 具体目标1 -基因递送：本目标的目标是构建、纯化和递送重组 腺病毒和慢病毒表达基因和小的抑制性RNA， 用于体内组织电穿孔的无内毒素质粒DNA。 具体目标2 -分析：该目标的目标是a）合成用于直接分析的重组蛋白质， 分析翻译后修饰和酶活性B）以提供以下的统一测量： 活性氧和氮类和c）确定蛋白质的亚细胞定位和鉴定蛋白质- 细胞内的共定位这些服务将由富尔顿博士（核心负责人）监督， 在重组病毒的生产和交付以及反应性分析方面具有丰富的经验， 氮物种和博士布莱克（共同核心领导人），谁是在表达和纯化的专家， 蛋白质，使用EPR技术来测量纯化蛋白质，细胞培养物和整个细胞中的ROS水平。 动物 相关性（见说明）： 这个核心的总体目标是提供一个集中的服务，促进基因的交付，通过 重组病毒和电穿孔，提供了活性氮的标准化测量， 生物体液中的活性氧，产生大量的重组蛋白和 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.