CORE--VECTOR PRODUCTION

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The Vector Core will play an integral and essential role in all three subprojects. First, all of the subprojects in this Program Project will require the availability of well characterized, high titer recombinant AAV (rAAV) 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 rAAV virus needed for all three subprojects in this Program. Each virus preparation will be purified by CsCl centrifugation and will be titered by a method (infectious center 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 and 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 physically removing contaminants in rAAV vector stocks. These will focus in particular on removing adenovirus contaminants using either ion exchange or antibody column chromatography. The core will also develop alternative adenovirus helpers which should eliminate the possibility of producing adenovirus contaminants in the rAAV vector stocks. The third goal of the Vector Core will be to develop improved marker genes and epitope tagged transgenes. The Vector Core will modify the green fluorescent gene b introducing specific mutations that increase the brightness and, therefore, the penetrance of the gene and they will construct versions of these marker genes that contain nuclear localization signals. The nuclear localized versions should make it easier to quantitate the frequency of transduction in a variety of in vivo experiments. Finally, the Core will construct epitope tagged versions of any neurotrophin or other therapeutic gene required by the subprojects in this Program.