Vector Core

在本申请的项目中提出的实验需要由各个实验室设计的质粒产生AAV载体。Vector Core Facility的目标是生产研究级AAV载体，用于在止血正常和血友病小鼠、非人灵长类动物和血友病犬中进行临床前研究。核心将确保重组AAV的可用性，用于表达各个项目所需的治疗性转基因或报告基因。AAV载体在无辅助病毒系统中产生，该系统基于使用提供AAV rep/cap和腺病毒辅助功能的两种辅助质粒和编码重组载体的第三种质粒对HEK-293细胞进行大规模转染。这将允许产生多种不同的载体（包括不同的转基因、表达载体、载体载体、载体、载体等）。 试剂盒和衣壳/血清型）供研究者使用。使用核心设施将提供可重现的载体产量和纯度，并且比在单个实验室生产载体更具成本效益，特别是对于涉及大型动物模型的实验。Core的服务将包括辅助质粒和载体质粒的大规模制备、使用磷酸钙沉淀法对HEK-293细胞进行大规模转染、通过细胞裂解回收和纯化重组AAV，然后进行差异沉淀和梯度离心、透析、无菌过滤和纯化载体的储存，以及定量狭缝印迹杂交。该中心将对载体制剂进行质量控制，并协助不同的项目进行功能测定。标准载体制备物预期产生约10[14]个载体。 每10个转瓶或等效的组织培养容器中的基因组。使用滚瓶方法的载体生产规模扩大已经得到优化（Biotechniques 34：1，2003），并将应用于大型动物研究的载体生产。核心将产生不同血清型的AAV载体，包括AAV-2和-8以及杂交载体。

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.