The Adenoviral Core (C) will provide necessary support for the creation, selection, characterization, production, and purification of the recombinant adenovirus required in subprojects 2, 3 and 4. The expertise for directing the core facility will be supplied by Mark Sussman, Ph.D.. This core will provide a variety of support services including: Production of the shuttle vector plasmid DNA (for distribution to investigators) and genomic DNA used for recombination experiments. 1. Maintenance of human embryonic kidney 293 cells permissive for adenoviral replication. 2. Cotransfection of transgene shuttle vector and adenoviral genomic DNA into 293 cells. 3. Monitoring cultures for DNA recombination resulting in adenoviral production. 4. Amplification of recombinant cultures. 5. Immunoblot assay to confirm expression of the transgene of interest (northern blots analyses will be used if antibodies are unavailable). 6. Clonal selection of confirmed recombinant viruses producing protein. 7. Purification and concentration of recombinant adenoviruses. 8. Titration of stocks for determination of plaque forming unit (pfu) levels. 9. Repository service for storage and cataloging of low passage viral stocks to serve as a backup for viral stocks given to investigators.Preparation of neonatal rat cardiomyocyte cultures.
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