1. Title: Cell Isolation, Culture and Protein Expression Core 2. Core Unit Leader: Paul, DiCorleto, Ph.D., Chair, Lerner Research Institute, Cleveland Clinic Technical Staff: Xiaolun Zhang, Alona Merkulova and Angela Money. 3. Description The purpose of this core is to provide cells and proteins as needed to the four Projects of the Program. The specific cells to be isolated by Core B personnel are: 1) blood cells, primarily platelets of human and murine origin;2) endothelial cells of human and murine origin;and 3) eukaryotic or prokaryotic cells expressing certain integrins, cytoskeletal proteins and mutants of these proteins. The prokaryocytic cells are used as a source for purified proteins;talin, kindlins and scr-family kinase derivatives, in the core and provide to the individual projects as needed. The preparation of cells by experience personnel will help to insure uniformity of purity and function. To have a reliable source of functional platelet preparations as needed by the projects is particularly valuable since the function of platelets can be highly variable. This is also true of endothelial cells of murine origin where experimental progress often depends on performing experiments with the cells derived from a small number of valuable mouse strains. The purified proteins are used in binding studies and for structural analyses, where relatively large quantities of high quality preparations are needed. With the need to prepare a limited number of proteins, isolation procedures can be standardized to provide high quality products. To perform these activities centrally will conserve resources, will help to insure quality and will allow for interproject comparison of results. In addition, since the individual Projects will still require substantial independent cell culture and will undoubtedly need to isolate cells for specific experiments, the Core will function in training members of the Program Project laboratories in proper cell culture and isolation methodologies and in appropriate ways to assess the quality of the cells. Thus, the Core serves an educational function as well. In the previous funding period, the Cell Culture and Expression were two separate entities. They are now consolidated into a single Core. This consolidation allows the new Core to provide more equitable service to all four Projects. Previously, Projects 2 made little use of the Cell Culture service and Projects 3 and 4 did not utilize the Protein Expression Core. Importantly, the consolidation also allows us to reduce the total annual direct costs from $250,426 for the prior Core B + C to $136,616 for the new Core B. This reduction is necessary because of annual budget rescissions and is feasible because the activities of the Core are conducted in adjacent space, allowing the core personnel to share responsibilities. Also, with the budgetary saving, we have been able to create a new In Vivo Core C ($111,900), which provides important services that will be used broadly in the Program. Dr. Paul DiCorleto serves as the Core Director. Dr. DiCorleto directed the Cell Culture Core within our program, which previously provided peripheral blood cells and endothelial cells to the Program Project. He is well-qualified to continue to lead this core and assume the additional responsibilities of the modified Core. While his role as LRI Chair is very time consuming, the technical staff is highly experienced and have been associated with the Cores of the Program Project for at least two years. While the core personnel will be trained to assist in all activities to meet demand, their primary activities are as follows: Angela Money isolates human platelets and endothelial cells;Alona Merkulova prepares murine platelets and endothelial cells;and Xiaolun Zhang expresses proteins in prokaryote and eukaryote cultures and isolates the expressed proteins. Mr. Zhang has more than 15 years experience in these activities. For convenience, the Core is located ion two rooms on NB2, the same floor as Dr. DiCorleto's office and lab and Dr. Qin's lab. One of these is equipped with tissue culture hoods and incubators, and the second has equipment for protein purification. Prioritization will be set routinely by Dr. DiCorleto. If demand for core activities is exceeding ability to supply, this will be discussed at our monthly Program Project meetings. Any unresolved priority decisions will be made by the Program Director.
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