CORE--VECTOR

载体核心的主要功能是提供重组AAV（rAAV）病毒原液并亚克隆本计划中使用的新质粒。Vector Core将使用经验证的质量控制试验，以确保项目中使用的rAAV制剂的纯度和比活性。此外，核心将实施纯化rAAV载体储备物的改进方法，并在重组AAV病毒可用时产生更高产量的重组AAV病毒。载体核心实验室将按照Meyer和King的项目所述制备高滴度浓缩rAAV血清型2、5或杂合5/2载体储备液。矢量将是 通过在UF部分由Core PI开发的无辅助质粒转染方法制备。将纯化rAAV载体，然后通过感染中心测定法对感染性病毒进行滴定，并通过狭缝印迹杂交对颗粒进行滴定。为了确定rAAV载体储备液的纯度，核心将对所有储备液进行银染色凝胶分析。复制型AAV（rcAAV）污染的程度（如有）将通过感染中心试验进行测定。

The Vector Core's primary function wili be to supply the recombinant AAV (rAAV)virus stocks and subclone new plasmids that are used in this Program. The Vector Core will use validated quality control assays to insure the purity and specific activity of the rAAV preparations used in the Program. In addition, the Core will implement improved methods for purifying rAAV vector stocks and generating higher yields of recombinant AAV virus at they become available. The Vector Core Laboratory will make high titer concentrated rAAV serotype 2, 5, or hybrid 5/2 vector stocks as described by the Projects by Meyer and King of this proposal. Vectors will be made by the helper free, plasmid transfection method developed at UF in part by the Core PI. rAAV vectors will be purified and then titered for infectious virus by the infectious center assay and for particles by slot blot hybridization. To determine the purity of the rAAV vector stocks, the Core will subject all stocks to silver stain gel analysis. The extent of replication competent AAV (rcAAV) contamination, if any, will be assayed by infectious center assay.