The aims of this Core continue to be to provide DDRCC Research Base investigators with: 1) a variety of cell lines of intestinal and hepatic origin; 2) expertise in specialized cell culture techniques; 3) expertise, protocols and reagents for retrovirus-mediated gene transfer and other techniques for manipulation of gene expression; 4) reagents and expertise for in vitro translation/translocation studies; 5) reagents and expertise for subcellular fractionation; and 6) expertise in studies of the biogenesis of polypeptides (i.e., pulse-chase radiolabeling and radioimmunoprecipitation techniques). The facility will maintain and store a variety of cell lines, and will provide a repository for stably transfected cell lines generated by Center members and for frozen morulae generated in the transgenic core. Fifteen cell lines are currently maintained by the facility and an additional eighteen are stored frozen. Plasmid DNA is available for fourteen vectors commonly used in transfection studies and others will be purchased as needed. A nominal fee will be charged for thawing frozen cell stocks, for specialized cell culture, and for aliquots of canine pancreatic microsomal vesicles. The Cell Biology Core will be under the supervision of Drs. Perlmutter (Director) and Teckman (Assistant Director), and will be established by expansion of an existing informal facility in Dr. Perlmutter's laboratory. Dr. Perlmutter will direct the overall program and supervise a technician, Natasha Simmons, who will be responsible for cell culture, expertise in retroviral-mediated gene transduction, and studies of biogenesis of newly synthesized polypeptides. Dr. Teckman, a new addition to the Core since the original submission of the application, will be responsible for in vitro translation/translocation studies and subcellular fractionation, and will supervise Pamela Hale in the maintenance of transfected cell lines and in providing training in transfection techniques. Drs. Jean Schaffer, Dan Ory, and Marc Levin will serve as an Internal Advisory Board for the Core. The facility is located in the Division of Gastroenterology and Nutrition in the Department of Pediatrics and is well-equipped for providing the proposed services. Three tissue culture hoods, three double incubators, water baths, a refrigerator and two large liquid nitrogen storage tanks are housed in the facility. A separate room is available for biohazard level two work and is designed for working with amphotrophic recombinant retroviral particles. Information regarding services and expertise provided by the facility will be disseminated by word-of-mouth, through a quarterly newsletter, in formal seminars (e.g., Gastroenterology research conference) sponsored by the Enrichment Program, and in a section of the annual Center newsletter. The Core will foster interaction and collaborations between Center members by: a) informing users of other investigators in the Center with similar projects and techniques, and b) through a quarterly newsletter provided via e-mail. Use of the facility will be restricted to members of the Center, investigators identified for recruitment to the Center, and investigators with P/F awards. All projects must be approved by the Core directors and Center Executive Committee. If necessary, the following prioritization scheme will be adopted: first priority will be given to regular users, followed by P/F investigators and junior investigators with limited funding, and then by collaborators in other fields selected for recruitment to the Center. Instruction and advice on cell culture techniques and various cell biological procedures will be available on request. Training will be done on a one-on-one basis and, when possible, will involve direct demonstration of the technique by the facility's staff. The Core staff will also be available for troubleshooting and help in designing modifications of standard protocols when necessary.
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