The Vector Core will play an integral and essential role in all subprojects. The primary mission of the Vector Core will to provide well characterized, high titer rAAV for the 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 other stocks regardless of the transgene or promoters used. In addition, quality control assays will be performed to assess the particle to infectivity ratio, general purity and the levels of wild- type AAV contamination and adenovirus contamination. This will involve the use of dot blot and/or PCR assays for particles, SDS-PAGE analysis of the stocks, as well as infectious center and plaque assays. The second goal of the Vector Core will be to develop new production methods for rAAV. Based on the discovery of the heparan sulfate proteoglycan as the AAV cell surface receptor, the core will focus on the use of heparan sulfate affinity column chromatography in the purification method. In addition, the Vector Core will further investigate the wild-type AAV helper functions in the development of better helpers to increase packaging efficiency of rAAV. Finally, the Vector Core will provide support in the construction of various vectors as needed by the subprojects of this Program. In particular, the Core will construct and test a series of CFTR minigenes under the control of selected constitutive promoters.
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