CORE--VECTOR PRODUCTION

向量核心将在所有这三个方面发挥不可或缺的重要作用 子项目。首先，此计划项目中的所有子项目将 需要特性良好、高滴度的重组AAV (RAAV)病毒库存。隔离这类股票是劳动密集型的，必须 以确保可重现质量的方式完成。因此， 载体核心的主要任务将是产生rAAV病毒 本计划中的所有三个子项目都需要。每种病毒制剂 将通过氯化铯离心法提纯，并将通过一种方法进行滴定 (感染中心分析)，这将允许与所有其他股票进行比较 不管使用的是转基因还是启动子。此外，质量 将进行对照试验以评估该颗粒的传染性。 每只鸡的比例和野生型AAV和腺病毒水平 污染。这将涉及使用斑点杂交和聚合酶链式反应分析 腺病毒、AAV和rAAV颗粒，以及感染中心检测 野生型AAV和腺病毒的空斑检测。这个 向量核心的第二个目标将是开发改进的方法 物理去除rAAV病媒种群中的污染物。这些将集中于 尤其是使用任一离子去除腺病毒污染物 交换层析或抗体柱层析。核心也将发展 替代腺病毒助手，应该消除了 在rAAV载体库中产生腺病毒污染物。第三 载体核心的目标将是开发改进的标记基因和 标记了表位的转基因。向量核心将修改绿色 荧光基因b引入特定的突变，增加了 亮度，因此，基因的外显性，他们将 构建这些包含核定位的标记基因的版本 信号。核本地化版本应该会使其更容易 不同体内信号转导频率的定量 实验。最后，核心将构建表位标记的版本 子项目所需的任何神经营养素或其他治疗性基因 这个节目。

The Vector Core will play an integral and essential role in all three subprojects. First, all of the subprojects in this Program Project will require the availability of well characterized, high titer recombinant AAV (rAAV) virus stocks. Isolation of such stocks is labor intensive and must be done in a manner that insures reproducible quality. Therefore, the primary mission of the Vector Core will be to generate the rAAV virus needed for all three subprojects in this Program. Each virus preparation will be purified by CsCl centrifugation and will be titered by a method (infectious center assay) that will allow comparison to all other stocks regardless of the transgene or promoters used. In addition, quality control assays will be performed to assess the particle to infectivity ratio of each stock and the levels of wild type AAV and adenovirus contamination. This will involve the use of dot blot and PCR assays for adenovirus, AAV, and rAAV particles, as well as, infectious center assays and plaque assays for wild type AAV and adenovirus, respectively. The second goal of the Vector Core will be to develop improved methods for physically removing contaminants in rAAV vector stocks. These will focus in particular on removing adenovirus contaminants using either ion exchange or antibody column chromatography. The core will also develop alternative adenovirus helpers which should eliminate the possibility of producing adenovirus contaminants in the rAAV vector stocks. The third goal of the Vector Core will be to develop improved marker genes and epitope tagged transgenes. The Vector Core will modify the green fluorescent gene b introducing specific mutations that increase the brightness and, therefore, the penetrance of the gene and they will construct versions of these marker genes that contain nuclear localization signals. The nuclear localized versions should make it easier to quantitate the frequency of transduction in a variety of in vivo experiments. Finally, the Core will construct epitope tagged versions of any neurotrophin or other therapeutic gene required by the subprojects in this Program.