CORE--VECTOR PRODUCTION

核心——矢量制作

• 批准号: 6565251
• 负责人: SERGEI ZOLOTUKHIN
• 金额: $ 24.29万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-12-01 至 2003-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6565251
• 关键词: adeno associated virus group; biomedical facility; genetic markers; microorganism culture; transfection /expression vector

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.

矢量核心将在所有三个方面发挥不可或缺的作用 次级项目。首先，本计划项目中的所有子项目将 需要获得充分表征的高滴度重组AAV （rAAV）病毒原种。隔离这些股票是劳动密集型的，必须 以确保可再现质量的方式进行。因此 载体核心的主要使命是产生rAAV病毒 这三个子项目都需要这个项目。各病毒制备液 将通过CsCl离心进行纯化，并通过一种方法进行滴定 （感染中心测定），可与所有其他储备液进行比较 而不管所用的转基因或启动子。此外，质量 将进行对照试验，以评估颗粒的感染性 每种原种的比例以及野生型AAV和腺病毒的水平 污染.这将涉及使用斑点印迹和PCR测定， 腺病毒、AAV和rAAV颗粒，以及感染中心测定 以及分别用于野生型AAV和腺病毒的噬斑测定。的 Vector Core的第二个目标是开发改进的方法， 物理去除rAAV载体储备物中的污染物。这些将集中在 特别是关于使用任一种离子去除腺病毒污染物 交换或抗体柱层析。核心也将发展 替代腺病毒助手，应该消除的可能性， 在rAAV载体储备物中产生腺病毒污染物。第三 载体核心的目标是开发改进的标记基因， 表位标记的转基因。矢量核心将修改绿色 荧光基因B引入特定的突变， 而他们，则是一群人，一群人， 构建这些标记基因的版本， 信号.核本地化版本应该更容易 定量各种体内转导频率 实验最后，核心将构建表位标记版本的 中的子项目所需的任何神经营养因子或其他治疗基因， 这个戒酒会这