CORE--VECTOR PRODUCTION

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The Vector Core will support all projects by providing expertise in the development, preparation, and characterization of oncoretroviral and lentiviral (HIV-1- and SIV-based) vectors. Murine leukemia virus (MuLV)-based oncoretroviral vectors will be produced through the generation of specific vector producer cells. A wide variety of packaging cells based on different envelope pseudotypes, including amphotropic, ecotropic, gibbon ape leukemia virus (GALV), vesicular stomatitic virus G (VSV-G) protein, and feline endogenous virus (RD114) are available, In some instances, vectors will also be generated through transient transfection of 293T cells with vector and packaging plasmids. Titers of vectors encoding the GFP marker will be determined by flow cytometric analysis for GFP expression in appropriate target cells, Accurate vector genome transmission without rearrangement will be confirmed by Southern blot analysis for all vectors, Vectors lacking GFP will be titered by Southem blot analysis using a probe derived from the packaging sequences which are universally present in all vectors, Vector producer cells will be tested for the presence of replication competent retrovirus using standard assays. The Core will also develop and prepare a variety of both HIV-1- and SIV-based vectors of self-inactivating (SIN) design using a 293T cell/four plasmid transfection method. Vectors with several different internal promoter elements are available. HIV-1- and SIV-based vector preparations will be titered using real time PCR for quantitiation of genome transmission into appropriate target cells. Concentration of vector preparations by both ultrafiltration and/or ultracentrifugation is available. Vector preparations are tested for the presence of replication competent lentivirus (RCL) using 1) an ELISA to assess the expression of gag antigen in passaged target cells and 2) an assay to test for the presence of transmitted and expressed tat. In ongoing and proposed work, the Core will characterize and evaluate whether non-replicative gag sequences are transmitted into target cells using our lentiviral vector systems. If needed, we will develop and test strategies to diminish this process, including using a codon-optimized gagpol expression plasmid. Additionally, the development of both HIV-1- and SIV-based pre-packaging, packaging, and vector producer cells will be a major goal of the Core in order to provide both higher titer and safer vectors.