This CORE will provide expertise in the preparation of both MuLV retroviral and lentiviral vectors and in the use of these vectors and in the use of these vectors in cell culture and animal model experimentation. MuLV vector preparations will be derived from stable producer cell clones or from 293T cells which have been transiently transfected with vector and packaging (helper plasmids). Titers of the conditioned medium from producer cell clones or 293T cells for vectors containing the GFP marker will be determined by FACS analysis of the transduced population of cells; e.g., ET3 or HeLa cells. In the context of these analysis, DNA will be harvested from cells which are 100% transduced and analyzed to evaluated transmission of the pro-viral vector genome using the packaging signal as a probe since these sequences will be present in all transmissible vector particles. Titers of vectors lacking the marker gene will be determined by Southern blot analysis of DNA from target cells transduced with various dilutions of the vector preparations. Lentiviral vectors are produced by co-transfection of 293T cells with a vector, GAG/POL helper, and ENV helper plasmids. Particles pseudotyped with the VSV-G envelope protein will be concentrated by ultra-centrifugation. Tittering is determined by transmission of the GFP marker of the intact pro-viral genome is determined by Southern blot analysis as described above. MuLV retroviral vector preparations are screened for replication competent retrovirus (RCR) by marker rescue from Mus. dunni cells. Lentiviral vector preparations are screened for RCR by measuring p24, a protein encoded by the GAG region, in exponentially growing lymphoid cells that are sensitive to infection by replication competent HIV. This CORE is responsible for maintaining supplies of cell lines, including retroviral producer clones, packaging cell lines as well as other lines that are required for the analysis of the vector preparations. The CORE will also be responsible for maintaining a computerized database for over 800 plasmids and for a variety of cell lines.
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