CORE--VECTOR FACILITY

核心——矢量设施

• 批准号: 6369122
• 负责人: NICHOLAS MUZYCZKA
• 金额: $ 18.75万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-25 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6369122
• 关键词: adeno associated virus group; affinity chromatography; biomedical facility; density gradient ultracentrifugation; genetic registry /resource /referral center; recombinant virus; transfection /expression vector; virus DNA

The Vector Core plays an integral and essential role in the production of research grade recombinant viral vectors for all of the investigators in the Program. It is critical that all investigators be provided with consistently high titer, pure rAAV preparations. It is particularly important that preps be free of replication-competent AAV (rcAAV) and have a particle to infectivity ratio of 200 or less. Therefore, the primary objective of the Vector Core is to make the highest quality vector possible for the investigators. Each virus preparation will be produced using a mini-Ad plasmid DNA system (to essentially eliminate wtAd contamination) and will be purified by a novel method utilizing iodixanol gradient centrifugation followed by heparin affinity chromatography. All virus stocks are subject to quality control assays to assess purity, particle titer, infectious titer, and potential contamination by rcAAV. The second objective of the Vector Core is to develop and implement improved methods in the process of scaling production and purification of rAAV. The Core is to develop and implement improved methods in the process of scaling production and purification of rAAV. The Vector Core is currently working on issues of scale-up production, including development of better helper systems. Additionally, we are testing new purification methods that include a variety of FPLC chromatography formats. Not only is the Vector Core able to make high quality rAAV virus preparations, but the Vector Core is also available to provide consulting on and/or actually perform vector construction and to aid in the development of biological assays is necessary for transgene screening. The Vector Core has already made available a variety of promoter-marker gene constructs for general screening.

载体核心在为该计划的所有研究人员生产研究级重组病毒载体方面发挥着不可或缺的重要作用。为所有研究人员提供一致的高滴度、纯 rAAV 制剂至关重要。尤其重要的是，制剂中不含具有复制能力的 AAV (rcAAV)，并且颗粒与感染率之比为 200 或更低。因此，Vector Core 的主要目标是为研究人员提供最高质量的矢量。每种病毒制剂都将使用微型 Ad 质粒 DNA 系统生产（以基本上消除 wtAd 污染），并通过利用碘克沙醇梯度离心和肝素亲和层析的新方法进行纯化。所有病毒库存均经过质量控制测定，以评估纯度、颗粒滴度、感染滴度和 rcAAV 的潜在污染。 Vector Core 的第二个目标是在 rAAV 的规模化生产和纯化过程中开发和实施改进的方法。核心是在rAAV的规模化生产和纯化过程中开发和实施改进的方法。 Vector Core 目前正在解决扩大生产的问题，包括开发更好的辅助系统。此外，我们正在测试新的纯化方法，包括各种 FPLC 色谱格式。 Vector Core 不仅能够制备高质量的 rAAV 病毒制剂，而且还可以提供有关载体构建的咨询和/或实际执行载体构建，并帮助开发转基因筛选所需的生物测定法。 Vector Core 已经提供了多种用于一般筛选的启动子-标记基因构建体。