The Viral Vector Core headed by Mark Sussman, Ph.D. will provide necessary support for the creation, selection, characterization, production, and purification of the recombinant adenoviruses required in the Program Project. Adenoviral vectors have become an accepted reagent for high efficiency introduction of foreign DNA into cardiomyocytes, which are refractory to conventional chemical methods of transfect ion. However, production of adenoviral vectors is a time consuming and technically complex task that is ideally suited for a core facility. The creation of a core will allow the principal investigators to be freed from the challenges of maintaining the high level of quality control that is essential for successful, rapid throughput recombinant adenoviral synthesis. This core will provide a variety of support services including: 1) production of the shuttle vector plasmid DNA (for distribution to investigators) and genomic DNA used for recombination experiments, 2) maintenance of human embryonic kidney 293 cells permissive for adenoviral replication, 3) cotransfection of transgene shuttle vector and adenoviral genomic DNA into 293 cells, 4) monitoring cultures for DNA recombination resulting in adenoviral production, 5) amplification of recombinant viral vectors, 6) immunoblot assay to confirm expression of the transgene of interest (northern blots analyses will be used if antibodies are unavailable), 7) clonal selection of confirmed recombinant viruses producing protein, 8) purification and concentration of recombinant adenoviruses, 9) titration of stocks for determination of plaque forming unit (pfu) levels, 10) repository service for storage and cataloging of low passage viral stocks to serve as a backup for viral stocks given to investigators.
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