CORE--VECTOR FACILITY

在为该计划中的所有研究人员生产研究级重组病毒载体的过程中，载体核心发挥着不可或缺的重要作用。至关重要的是，向所有研究人员提供持续的高滴度、纯rAAV制剂。特别重要的是，PREPS不含复制能力AAV(RcAAV)，并且具有200或更低的颗粒与传染性比率。因此，向量核心的首要目标是为调查人员提供尽可能高质量的向量。每一种病毒制剂都将使用迷你Ad质粒DNA系统(基本上消除wtAd污染)生产，并将通过一种新的方法进行纯化，该方法使用碘二醇梯度离心法，然后使用肝素亲和层析。所有病毒库存都要经过质量控制分析，以评估纯度、颗粒滴度、感染性滴度和可能受到rcAAV的污染。载体核心的第二个目标是在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。向量核心目前正在研究扩大生产的问题，包括开发更好的助手系统。此外，我们正在测试新的纯化方法，包括各种FPLC层析格式。载体核心不仅能够制作高质量的rAAV病毒制剂，而且还可以提供载体构建的咨询和/或实际执行载体构建的咨询，并帮助开发转基因筛选所必需的生物检测方法。载体核心已经提供了各种启动子-标记基因结构用于一般筛选。

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.