The Gene Transfer Core (GTC) supports gene transfer studies in Projects 1,2 3 and 4. These projects are? highly dependent on achieving efficient gene transfer in primary cells, including hematopoietic progenitor? cells, T lymphocytes and dendritic cells. In the upcoming grant cycle, the GTC will mainly focus on the? development and optimization of clinical processes and on the implementation of the clinical trials.? The specific aims of the Gene Transfer Core are to carry out and/or coordinate: 1. Generation and? characterization of high-titer producer cell clones, master cell banks (MCB) for clinical studies 2. Production? and titration of 5 to 15 liter batches of clinical viral stocks in semi-closed systems 3. Production and biosafety? testing of clinical grade plasmid DNA vaccine for immunization 4. Expansion and transduction of patient cells? in semi-closed systems in collaboration with the investigators of each clinical trial 5. Detection of replicationcompetent? retrovirus and other biosafety testing in cultured packaging cell clones (MCB), viral stocks and? clinical specimen 6. Generation and biosafety testing of clinical AAPCs 7. Detection of oncoretroviral vector? integration sites by LM-PCR in patient cells 8. Cell banking, storage of viral stocks, plasmid DNA vaccine? and clinical specimens. In addition, the GTC provides essential advisory and training functions for the? generation of research grade reagents within the program. The Projects are thus advised or trained on 1.? How to construct recombinant oncoretroviral- and lentiviral- vectors, plasmid DNA vectors, siRNA encoding? retroviral vectors 2. What packaging cell lines to use 3. How to transfect vector DNA in packaging cells and? select producer cell lines 4. What tests to perform to analyze gene expression 5. How to titrate cell-free? retroviral stocks by flow cytometry, Southern blot or real time PCR analysis.? The GTC is also a repository of numerous reagents and protocols that are freely distributed to all members? of the Program who request them. A list of these reagents and protocols will be posted on the newly created? Program web portal to facilitate distribution and interactions between the Projects. Vector production,? plasmid DNA manufacturing and cell transduction in the GTC decreases the cost of clinical development,? ensures high quality and consistency of molecular and cellular processes, and their availability to all? investigators in the Program.?
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