Gene Vector and Production Core1. Core Function:This viral vector core function is to provide each of the four projects two major services:(1) Design and construction of transgenes for packaging within viral vectors;(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 pseudotyperecommendations, 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, andthen appropriately tested for transgene expression.Production, purification and testing include:A) Production of:AAV serotypes 1 -9Adenovirus type 2VSVG pseudotyped LentivirusB) Purification will utilize cesium chloride gradient centrifugation for AAV and Adenovirus and sucrose cushiongradients for Lentivirus production, check post production purification schemes.C) Testing of each virus that will include:i) Titration of the viruses after dialysis prior to delivery to project leaders.ii) Testing of the in vitro transduction efficiency of viruses in HeLa cells for CMV or other strongpromoters, or primary neonatal rat cardiomyocytes for restricted (cardiac) promoters.iii) Western blot analysis will be used to establish expression of viral transgenes prior to delivery toproject 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 (100s of 15-cm plates) production of specific transgenesas dictated by PPG participant needs. We will provide the molecular biological support related to sub-cloningof novel therapeutic genes, inhibitory and micro RNAs, as well as enhancer-promoter configurations for viraldevelopment as needed by the Project Leaders. The main functions of Core D are; 1) Production of AdenoandAdeno-associated viruses, 2) Purification of these viruses, and 3) Testing viruses prior to transferring tomembers of the Scientific Program. An additional function will be the production of VSVG pseudotypedlentivirus, as needed by members of the Scientific Program.
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