The Molecular Biochemical and Anatomical Cell Culture Core Laboratory will provide central support to all the projects in the Program. This Core facility will evolve from an already established Core Laboratory which has been part of the Center for Neurodegenerative Diseases. The Core already has extensive capabilities to support histological and molecular biological methods, including qualitative and quantitative in situ hybridization at both the film and emulsion levels, electron microscopy, immunohistochemistry, 3-dimensional anatomical reconstruction (Eutectic), and RT-PCR. Because of the extensive use of cultured human cells in this Program, it will be necessary for the Core to add centralized cell culture facilities to provide cultured human cells in this Program, it will be necessary for the Core to add centralized cell culture facilities to provide for the efficient use of these cells in the four Projects. The Core will be directed by Patricia Trimmer, Ph.D. Dr. Trimmer is an accomplished neuroscientist who already has technical capabilities in all areas of Core support. She will function practically as a Co-Investigator on all three Project, in addition to being P.I. on her own project, and will supervise a Laboratory Specialist Sr. Dr. Parker and Janice Parks (Project 1) will contribute to the Core Lab and participate in the characterization, maintenance and storage of the over 100 cybrid cell lines currently available Together these individuals will interact with all four projects in the following manner: Project 1. Dr. Parker's group will generate the cybrid cell lines from patients' platelet mitochondria and Rho/o SY5Y neuroblastoma and NT2, but these cell lines will be maintained and stored in the Core. Dr. Trimmer will work with Dr. Parker to define anatomically the dynamics of mitochondrial transfer and survival in the cybrid cells. The Core will also be responsible for establishing clonal lines of cybrid cells and will make available to all PI's the different cell lines. Project 2. The Core will provide differentiated cybrids and primary neurons for Dr. Trimmer to use in her studies of mitochondrial dynamics. Project 3. The Core will serve as a source of multiple cybrid cell lines and primary neurons for Dr. Tuttle's group, and will also provide a physical location for some of Dr. Tuttle's experiments to be carried out. Drs. Trimmer and Tuttle will collaborate in establishing biochemical and histological signs of apoptosis in the cybrids. Project 4. The Core will provide multiple cybrid cell lines to Dr. Bennett's group to measure changes in both oxygen free radicals and expression of growth regulatory genes during exposure to mitochondrial toxins. The Core will also support the RT-PCR, histological and in situ hybridization studies in Project 4. Because all cybrid cell lines will be centrally inventoried in the Core, coordination among all PI's as to which individual cell lines are being studied will insure comparability of data. The Core will also have a computing facility to allow efficient record keeping of data collected on cybrids by the PI's.
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