Gene Vector and Production Core

1.核心功能： 这个病毒载体的核心功能是为四个项目中的每一个提供两大服务： (1)用于病毒载体内包装的转基因的设计和构建; (2)这些病毒载体类型的生产、纯化和测试。 包装质粒的设计和构建包括： A）每种病毒载体都有特定的设计要求，包括包装容量、血清型和假型 建议，这些将在构建每个新的包装质粒之前进行检查。 B）通过限制性内切酶和测序确认每个包装质粒的构建，和 然后适当地测试转基因表达。 生产、纯化和检测包括： A.生产： AAV血清型1 - 9 2型腺病毒 VSVG假型慢病毒 B）纯化将利用氯化铯梯度离心用于AAV和腺病毒以及蔗糖垫 对于慢病毒生产的梯度，检查生产后纯化方案。 C）测试每种病毒，包括： i）在透析后，在交付给项目负责人之前，对病毒进行滴定。 ii）测试病毒在HeLa细胞中对CMV或其他强病毒的体外转导效率 启动子，或限制性（心脏）启动子的原代新生大鼠心肌细胞。 iii）蛋白质印迹分析将用于在递送至 项目负责人。 该核心将生产和纯化AAV血清型1-9、腺病毒2型和慢病毒。具体地说， 我们将开发和维持用于大规模生产特定转基因的细胞系（100个15 cm平板）。 根据PPG参与者的需求决定。我们将提供与亚克隆相关的分子生物学支持 新的治疗基因，抑制性和微小RNA，以及病毒的增强子-启动子构型， 根据项目领导人的需要。核心D的主要功能是：1）腺病毒的生产 腺相关病毒，2）这些病毒的纯化，和3）在转移到 科学计划的成员。一个额外的功能将是VSVG伪类型的生产 慢病毒，如科学计划成员所需。

1. Core Function: This viral vector core function is to provide each of the four projects two major services: (1) Design and construction of transgenes for packaging within viral vectors; (2) Production, purification and testing of those viral vector types. Design and construction of packaging plasmids include: A) Each viral vector has specific design requirements including packaging capacity, serotype and pseudotype recommendations, these will be examined prior to construction of each new packaging plasmid. B) The construction of each packaging plasmid will be confirmed by restriction digests and sequencing, and then appropriately tested for transgene expression. Production, purification and testing include: A) Production of: AAV serotypes 1 -9 Adenovirus type 2 VSVG pseudotyped Lentivirus B) Purification will utilize cesium chloride gradient centrifugation for AAV and Adenovirus and sucrose cushion gradients for Lentivirus production, check post production purification schemes. C) Testing of each virus that will include: i) Titration of the viruses after dialysis prior to delivery to project leaders. ii) Testing of the in vitro transduction efficiency of viruses in HeLa cells for CMV or other strong promoters, or primary neonatal rat cardiomyocytes for restricted (cardiac) promoters. iii) Western blot analysis will be used to establish expression of viral transgenes prior to delivery to project leaders. This core will manufacture and purify AAV serotypes 1-9, Adenovirus type 2 and Lentivirus. Specifically, we will develop and maintain cell lines for large scale (100s of 15-cm plates) production of specific transgenes as dictated by PPG participant needs. We will provide the molecular biological support related to sub-cloning of novel therapeutic genes, inhibitory and micro RNAs, as well as enhancer-promoter configurations for viral development as needed by the Project Leaders. The main functions of Core D are; 1) Production of Adenoand Adeno-associated viruses, 2) Purification of these viruses, and 3) Testing viruses prior to transferring to members of the Scientific Program. An additional function will be the production of VSVG pseudotyped lentivirus, as needed by members of the Scientific Program.