The Vector Core will play an integral and essential role in all subprojects. First, all of the Subprojects in this Program Project will require the availability of well characterized, high titer recombinant virus stocks. Isolation of such stocks is labor intensive and must be done in a manner that insures reproducible quality. Therefore, the primary mission of the Vector Core will be to generate the recombinant virus needed for the Subprojects in this Program. Two kinds of recombinant virus will be available, Adeno-associated virus (AAV) and Adenvirus. Each virus preparation will be purified by CsC1 centrifugation and will be titered by a method (infectious center assay, plaque assay) that will allow comparison to all other stocks regardless of the transgene or promoters used. In addition, quality control assays will be performed to assess the particle to infectivity ratio of each stock and the levels of wild type AAV or replication competent adenovirus contamination. This will involve the use of dot blot and PCR assays for adenovirus, AAV, and rAAV particles, as well as, infectious center assays and plaque assays for wild type AAV and adenovirus, respectively. The second goal of the Vector Core will be to develop improved methods for preparing rAAV stocks. These will focus inparticular on developing herpesvirus helper viruses carrying the AAV rep and cap genes which show promise of increasing the yield of rAAV virus preparations when used in conjunction with producer cell lines carrying rAAV proviruses. The third goal of the Vector Core will be to develop improved methods for construction of adenovirus recombinants using the Cre-Lox P vectors that can be assembled in vitro prior to the growth of recombinant adenovirus. Finally, the Vector Core will introduce methods for the growth of improved lentivirus vectors described in Subproject 4 (Chang) when they become available.
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