CORE--VECTOR

载体核心的主要功能将是提供重组AAV(RAAV)病毒库，并亚克隆本计划中使用的新质粒。载体核心将使用经过验证的质量控制分析，以确保该计划中使用的rAAV制剂的纯度和比活性。此外，Core将实施改进的方法来纯化rAAV载体库存，并在重组AAV病毒可用时产生更高产量的重组AAV病毒。病媒核心实验室将按照迈耶和金在本提案中的项目描述的那样，制作高滴度的浓缩rAAV血清型2、5型或混合5/2病媒种群。向量将是 由UF发展起来的无辅助子的质粒转染法，部分由Core PI制成。RAAV载体将被纯化，然后用感染中心法检测感染性病毒，用狭缝杂交检测颗粒。为了确定rAAV载体库的纯度，Core将对所有库进行银染凝胶分析。复制能力AAV(RcAAV)的污染程度，如果有的话，将通过感染中心试验进行检测。

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.