The application is for a Shared Instrument Grant (SIG) to purchase a new flow cytometer for the Genetic, Immunologic and Stem Cell Therapy (GIST) program at Childrens Hospital Los Angeles (CHLA). The GIST program consists of 6 NIH-funded principal investigators who perform basic, translational, and clinical research. Over 25 post-doctoral fellows, graduate students, and technicians perform studies that are heavily dependent on FACS for analyses and isolation of cells. Because flow cytometry has been a key methodologic tool for the research performed by the GIST investigators, the program has built its own FACS Core Laboratory over the last 12 years to support its research. The present Core includes two analytic cytometers and one sorter, which are housed in a dedicated FACS laboratory and supported by 2 FTE cytometry technicians. Gene transfer studies that are performed include analyses of gene transfer into cells from both preclinical models (murine, chimeric mouse/human, and canine models) as well as analyses and sorting of cells from patients participating in clinical gene transfer trials. The preclinical studies depend on multiparametric flow cytometry with both monoclonal antibody-based and enhanced green fluorescent protein (EGFP)- based labeling of transduced cells. Future studies are expected to increasingly employ competitive repopulation assays in which vectors containing different EGFP variants will be used to mark cells transduced with different vector designs or under different conditions. Immunoloqic studies performed in the program are mainly focused on studies of the normal ontogeny of lymphocytes, lymphocyte development after hematopoietic stem cell transplantation (HSCT), and the function of cytokine receptors in lymphopoiesis. Again, these studies depend on combined antibody and EGFP-based assays as well as nucleic acid staining to measure proliferation and/or apoptosis. Analyses of receptor function are increasingly focused on techniques that employ fusion proteins of EGFP spectral variants and receptors; these studies depend on FACS isolation of cells using a broad spectrum of colors for excitation and emission. The stem cell studies performed by members of the program are basic biologic studies of immunophenotypically characterized hematopoietic progenitors, and increasingly, progenitors of other lineages including mesenchymal and epithelial cells. The stem cell studies also depend on multiparametric studies with antibodies, EGFP, and nucleic acid staining, and complex analyses of progeny that develop in vivo in chimeric mice as well as in vitro cultures. The present grant is to purchase a new cell sorter to increase the GIST program's productivity. The sorter will provide additional analytic and sorting capabilities, which the present equipment cannot support. The new sorter will also allow the program to grow by providing additional sort time for the increasing number of GIST program investigators, thus preventing FACS availability from becoming a research bottleneck. The new sorter will be configured for maximal spectral range, flexibility and speed of cell sorting, and sterility of sorted cells. In order to accommodate the new sorter, the FACS Core Laboratory will be remodeled and expanded with institutional funds. Combined institutional and grant support will be used to staff and maintain the new equipment.
