Vector Core

矢量核心

• 批准号: 7155001
• 负责人: Roland W. Herzog
• 金额: $ 32.16万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-29 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7155001
• 关键词: adeno associated virus group; biomedical facility; biotechnology; cooperative study; hemophilia As; hemophilia B; plasmids; recombinant proteins; transfection /expression vector

Experiments proposed in the projects of this application require production of AAV vectors from plasmids designed by the individual laboratories. The goal of the Vector Core Facility is the production of research grade AAV vector for pre-clinical studies in hemostatically normal and hemophilic mice, non-human primates, and in hemophilic dogs. The Core will ensure the availability of recombinant AAV for expression of therapeutic transgenes or reporter genes as required by the individual projects. AAV vector is produced in a helper virus-free system based on large-scale transfection of HEK-293 cells using two helper plasmids that supply AAV rep/cap and adenoviral helper functions and a third plasmid encoding the recombinant vector. This will allow production of a variety of different vectors (including different transgenes, expression cassettes and capsids/serotypes) for the investigators. The use of a Core Facility will provide reproducible yield and purity of vector, and will be more cost-effective than vector production in individual laboratories, in particular for experiments that involve large animal models. The service of the Core will include large-scale preparation of helper and vector plasmids, large-scale transfection of HEK-293 cells using calcium phosphate precipitation, recovery and purification of recombinant AAV by cell lysis followed by differential precipitation and gradient centrifugation, dialysis, sterile filtration, and storage of purified vector, and quantitative slot blot hybridization. The Core will carry out quality control of vector preparations and assist the different projects with functional assays. Standard vector preparations are expected to yield about 10[14] vector genomes per 10 roller bottles or equivalent tissue culture vessels. Scale-up of vector production using a roller bottle method has been optimized (Biotechniques 34:1, 2003) and will be applied to production of vector for large animal studies. The Core will produce AAV vector of different serotypes including AAV-2, and -8, and hybrid vectors.

在本申请项目中提出的实验需要从单个实验室设计的质粒中生产AAV载体。矢量核心设施的目的是生产研究级AAV载体，用于血压正常和血友病小鼠，非人类灵长类动物以及血友病犬的临床前研究。核心将确保根据单个项目的要求，可用性AAV可用于表达治疗转基因或报告基因的基因。 AAV载体是在无辅助病毒系统中基于对HEK-293细胞进行大规模转染的两个辅助质粒的大规模转染的产生的，这些助手质粒提供AAV REP/CAP/CAP和腺病毒辅助功能以及编码重组载体的第三个质粒。这将允许产生各种不同的向量（包括不同的转基因，表达式 研究人员的盒式胶囊和衣壳/血清型）。核心设施的使用将提供可再现的载体和纯度，并且比单个实验室中的矢量产生更具成本效益，尤其是对于涉及大型动物模型的实验。核心的服务将包括大规模制备辅助器和载体质粒，使用磷酸钙沉淀，通过细胞裂解进行重组AAV的大规模转染HEK-293细胞，然后进行差分降水和梯度偏心，透析，透析，透析，透析无菌过滤和纯化载体的存储以及定量插槽印杂交。核心将对矢量制备进行质量控制，并通过功能测定协助不同的项目。标准矢量制剂预计将产生约10 [14]矢量 每10个滚筒瓶或等效组织培养容器的基因组。使用辊瓶方法对矢量产生的扩展已得到优化（生物技术34：1，2003），并将用于大型动物研究的载体的生产。核心将产生不同血清型的AAV载体，包括AAV -2和-8和混合矢量。