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.
(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.
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