Adenovirus Collection - Newgard Subcollection
The BCBC Adenovirus Collection is a repository of replication-deficient recombinant adenoviruses made and/or accumulated and maintained by the laboratory of Dr. Chris Newgard at the Sarah W. Stedman Nutrition and Metabolism Center, Duke University Medical Center in Durham, North Carolina. The repository is accessible through this searchable database, created and maintained by the BCBC Coordinating Center, and through which individual viruses may be requested. Upon request, the user will receive an aliquot of viral stock that may be used for further amplification in HEK293 cells. HEK293 cells are available though the ATCC, or from the investigator's own institutional tissue culture core facility, if available.
Recombinant adenoviruses have long been used as efficient gene-delivery vehicles into a wide range of hard-to-transfect cells, including, but not limited to islet beta cells and beta-cell-derived cell lines, primary hepatocytes, neuronal cells, and myocytes. For a review of adenoviral biology and applications, see the reviews by Amafitano, 2004 and Antinozzi and Newgard, 1999. The viruses available through the BCBC are largely first-generation (E1-deleted) viruses that have been engineered for the overexpression of cloned cDNAs or for RNAi-mediated suppression of gene expression. Classes of cloned cDNAs and RNAi targets include transcription factors, metabolic enzymes, receptors, signaling molecules, and secreted peptides. In addition, a number of control viruses are available, including viruses that contain the b-gal or EGFP reporter genes.
Currently, overexpression viruses utilize the CMV promoter for high-level constitutive expression in all cell types. A beta-cell-specific viral system which utilizes the rat insulin I promoter (RIP) to drive the expression of cloned cDNAs is currently being developed and tested. As these new viruses are generated, they will be made available as well.
The RNAi adenoviruses available through the BCBC repository largely utilize the human HI promoter to drive siRNA expression in a non-cell-type-specific and constitutive manner. A beta-cell-specific adenoviral RNAi system, utilizing the RIP to drive expression of the siRNA is currently also being developed; like new overexpression viruses, these will be made available as they are generated and validated.
Finally, whether published or unpublished, the viruses listed have been shown to overexpress or silence their respective genes. The RNAi viruses generally reduce target gene expression by 60-90%. The precise levels of expression or silencing achieved by an investigator in any particular experiment, however, is a function of cell type, transduction conditions, protein stability, and time course. While some transduction guidelines exist, investigators should be prepared for some optimization, if warranted.