CORE--VECTOR

向量核心将在所有 子项目。向量核心的主要任务是提供良好的 为本计划中的子项目提供特性化、高滴度的rAAV。每个人 病毒制剂将通过Cscl离心法提纯，并将被 通过一种允许比较的方法(感染中心分析)进行滴定 转移到其他种群，而不考虑使用的转基因或启动子。在……里面 此外，将进行质量控制分析，以评估 颗粒物对感染力的比率、总纯度和野生- AAV污染类型和腺病毒污染类型。这将涉及到 使用斑点杂交和/或聚合酶链式反应分析颗粒，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析 以及传染病中心和斑块分析。这个 向量核心的第二个目标将是开发新的生产方法 用于rAAV。基于硫酸乙酰肝素蛋白多糖AS的发现 AAV细胞表面受体的核心将重点放在肝素的使用上 硫酸盐亲和柱层析中的纯化方法。在……里面 此外，媒介核心将进一步调查野生型AAV 帮手功能在开发中更好地增加帮手 重组AAV的包装效率。最后，向量核心将提供 根据需要支持各种载体的构建 本计划的子项目。特别是，核心将构建和 在选定的控制下测试一系列CFTRMiniges 构成推动者。

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.