CORE--VECTOR

核心--向量

• 批准号: 6318400
• 负责人: SERGEI ZOLOTUKHIN
• 金额: $ 27.1万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6318400
• 关键词: adeno associated virus group; affinity chromatography; biomedical facility; biotechnology; chloride channels; heparan sulfate; technology /technique development; transfection /expression vector

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.

矢量核心将在所有领域发挥不可或缺的重要作用。 次级项目。向量核心的主要使命是 本项目中的子项目的特征性高滴度rAAV。每个 将通过CsCl离心纯化病毒制剂， 通过允许比较的方法（感染中心测定法）进行滴定 与其它原种无关，而与所用的转基因或启动子无关。在 此外，将进行质量控制测定，以评估 颗粒与感染性比率、一般纯度和野生- 型腺相关病毒污染和腺病毒污染。这将涉及 颗粒的斑点印迹和/或PCR分析的使用，SDS-PAGE分析 以及感染中心和噬菌斑测定。的 Vector Core的第二个目标是开发新的生产方法 对于rAAV。基于硫酸乙酰肝素蛋白聚糖作为 AAV细胞表面受体，核心将集中在使用乙酰肝素 纯化方法中的硫酸盐亲和柱层析。在 此外，载体核心将进一步研究野生型AAV helper函数的开发，更好的增加helper rAAV的包装效率。最后，Vector Core将提供 支持建设各种载体的需要， 该项目的子项目。特别是，核心将建设和 在选择的控制下测试一系列CFTR小基因， 组成型启动子