The Retroviral Core is designed to support gene transfer studies in Projects. These projects are high dependent on achieving efficient retroviral-mediated gene transfer in primary cells of hematopoietic origin, including progenitor cells and T lymphocytes. The proposed studies all require well-characterized and periodically tested high-titer producer cell lines and cell-free retroviral stocks, as well as systematic detection of replication-competent retrovirus (RCR). The centralized generation of high-quality producer cell lines and retroviral stocks and biosafety testing will be cost effective, permit standardization of procedures, and facilitate the exchange of information and expertise between the three projects. In the pre-clinical phase of investigation, the specific aims of the Retroviral Core are: 1. Construction of the recombinant retroviral vector; 2. Transfection of vector DNA in packaging cell lines and selection of producer cells; 3. Characterization of vector transmission in standard cell lines by Southern blot analysis; 4. Expansion of independent clones and identification of the best clone for the intended target cells; 5. Viral titration of cell-free supernatant; and 6. Detection of RCR in cell-free viral stocks and transduced target cells. The construction and analysis of retroviral vectors, the generation and characterization of producer cell lines, and the detection of RCR are carried out in the laboratory of Dr. M. Sadelain. In the clinical phase of investigation, the specific aims of the Retroviral Core are to carry out and/or coordinate: 1. the generation of high-titer producers and retroviral stocks for clinical studies; 2. the detection of RCR in cultured cells and clinical specimens; and 3. cell banking and storage of viral stocks. While certain test that are integral to retroviral production for clinical purposes will be contracted to extramural facilities, our ability to execute most of the production and testing in a new purpose-built Clinical Gene Transfer Facility will greatly decrease the cost of generating high-quality material and, therefore, the cost of clinical investigation. M. Sadelain, M.D., Ph.D., is the principal investigator in the Retroviral Core, assisted by M. Gallardo, M.Sc., and C. Tan, M.D., M.Sc., two senior research assistants with considerable experience in tissue culture and in molecular biology, respectively. One additional full-time technical person will be needed for the generation and characterization of producer cell lines. One investigator will be recruited for supervising all technical procedures related to clinical investigation.
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