CORE--VECTOR FACILITY

载体核心在为该项目的所有研究者生产研究级重组病毒载体方面发挥着不可或缺的作用。至关重要的是，向所有研究者提供一致的高滴度纯rAAV制剂。特别重要的是，制备物不含可复制的AAV（rcAAV），并且具有200或更小的颗粒与感染性比率。因此，Vector Core的主要目标是为研究者提供最高质量的载体。将使用mini-Ad质粒DNA系统（以基本消除wtAd污染）生产每种病毒制备物，并将通过利用碘克沙醇梯度离心然后肝素亲和层析的新方法进行纯化。对所有病毒原液进行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目前正在研究扩大生产的问题，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Vector Core还可用于提供咨询和/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。载体核心已经提供了各种启动子标记基因构建体用于一般筛选。

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.