CORE--IMMUNE

本申请的4个项目中提出的大多数实验都需要从 个体研究人员使用标准的重组DNA技术设计的质粒。的目标是 因此，载体核心设施是用于临床前研究的研究级AAV载体的生产 止血正常和血友病小鼠以及非人类灵长类动物。此功能将确保 重组AAV在涉及AAV载体实验的不同项目中的可用性 治疗性转基因的表达。AAV载体是在一个基于大规模的辅助病毒无病毒系统中产生的 AAV rep/Cap和AAV-Rep/Cap两种辅助性载体对HEK-293细胞的转染作用 腺病毒辅助功能和编码重组载体的第三质粒。这将允许生产 为调查人员提供了各种不同的媒介。核心设施的使用将提供可重复的收益 和媒介的纯度，并将比在个别实验室生产媒介更具成本效益，在 尤其是涉及大型动物模型的实验。核心的服务将包括大规模的 辅助性和载体载体的制备及钙离子大规模转染HEK-293细胞 细胞裂解差示磷酸盐沉淀法回收纯化重组AAV 沉淀和梯度离心、透析、无菌过滤和纯化载体的储存，以及 定量缝隙印迹杂交。核心将指导质量控制的准备工作，并协助 不同的项目与功能分析。标准载体制剂预计可产生约10(13)个载体 虽然扩大规模将导致每次制备大约10(14)个载体基因组。纵向扩展 使用滚筒瓶法的媒介生产已得到优化，并将 用于生产用于非人灵长类研究的载体。核心还将扩展和净化 重组腺病毒载体，并存储和分发用于免疫学分析的多肽库。

Most experiments proposed in the 4 projects of this application require production of AAV vector from plasmids that the individual researchers design using standard recombinant DNA techniques. The goal of the Vector Core Facility is therefore the production of research grade AAV vector for pre-clinical studies in hemostatically normal and hemophilic mice and in non-human primates. This function will ensure the availability of recombinant AAV for the different projects that involve experiments with AAV vectors for expression of therapeutic transgenes. AAV vector is produced in a helper virus-free system based on largescale plasmid 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 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 d fferential precipitation and gradient centrifugation, dialysis, sterile filtration, and storage of purified vector, and quantitative slot blot hybridization. The Core will vector preparations for quality control and assist the different projects with functional assays. Standard vector preparations are expected to yield approximately 10(13) vector genomes, while scale up will result in production of approximately 10(14) vector genomes per preparation. Scale-up of vector production using a roller bottle method has been optimized and will be applied to production of vector for non-human primate studies. The Core will also expand and purify recombinant adenoviral vectors and store and distribute peptide libraries for immunological assays.