This viral vector core function is to provide each of the three scientific projects two major services: 1. Design and construction of enhancer/promoters and transgenes for packaging within viral vectors;and 2. Production, purification and testing of those viral vector types. Design and construction of packaging plasmids include: A) Each viral vector has specific design requirements including packaging capacity, serotype and pseudotype recommendations, these will be examined prior to construction of each new packaging plasmid. B) The construction of each packaging plasmid will be confirmed by restriction digests and sequencing, and then appropriately tested for transgene expression. Production, purification and testing include: A) Production of: AAV serotypes 1-9, Adenovirus type 5 and pseudotyped Lentivirus B) Purification of: AAV will depend on serotype. AAV serotypes 1-9 have been purified by iodixanol gradient centrifugation then fast protein liquid chromatography (FPLC), followed by dialysis. For viral titration methods we use transgene primers and real-time PCR to quantify the titer of the virus. To examine the purity of the virus we use silver staining of SDS polyacrylamide gels. To test for biological contaminants we add aliquots of the purified virus to cells in culture without antibiotics. Experience has demonstrated that if the number of viral particles obtained from each cell is less than 5000 for AAV then the prep will be discarded. Purification of: Adenovirus we use the Adenopure columns supplied by Puresyn, Inc. (Malvern, PA) the titers have been comparable to titers using cesium. Purification of: Lentivirus continues to utilize sucrose cushion gradients. In addition there are a number of commercially available lentivirus concentration and purification kits (Cell Biolabs;San Diego, CA). C) Testing of each virus that will include: 1) Titration of the viruses after dialysis prior to delivery to project leaders. 2) Testing of the in vitro transduction efficiency of viruses in HeLa cells for CMV or other strong promoters, or primary neonatal rat cardiomyocytes for restricted (cardiac) promoters. 3) Western blot analysis will be used to establish expression of viral transgenes prior to delivery to project leaders. This core will manufacture and purify AAV serotypes 1-9, Adenovirus type 2 and Lentivirus. Specifically, we will develop and maintain cell lines for large-scale production of specific vectors and transgenes as dictated by PPG participant needs. We will provide the molecular biological support related to sub-cloning of novel therapeutic genes, inhibitory and micro RNAs, as well as enhancer-promoter configurations for viral development as needed by the Project Leaders. The main functions of Core D are;1) Production of Adenoand Adeno-associated viruses, 2) Purification of these viruses, and 3) Testing viruses prior to transferring to members of the Scientific Program.
The Viral vector core supports all four scientific projects providing both large and small-scale production expertise for multiple viral platforms. Having packaging plasmids with unique restriction enzymes for new transgene cloning provides an important starting point for virus production. We have developed multiple packaging plasmids containing strong viral and cardiac biased enhancer/promoter configurations that have been shown to express recombinant transgenes.
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