Gene Vector Core

基因载体核心

• 批准号: 7488131
• 负责人: JOSEPH E RABINOWITZ
• 金额: $ 21.18万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7488131
• 关键词: Adenoviruses; Biological; Cardiac; Cardiac Myocytes; Cell Line; Centrifugation; Cytomegalovirus; Dependovirus; Development; Dialysis procedure; Enhancers; Facility Construction Funding Category; Genes; Hela Cells; In Vitro; MicroRNAs; Molecular; Neonatal; Participant; Physical Dialysis; Plasmids; Production; Rattus; Recommendation; Scheme; Serotyping; Services; Subfamily lentivirinae; Sucrose; Testing; Titrations; Transgenes; Viral; Viral Vector; Virus; Western Blotting; cesium chloride; design; design and construction; member; novel therapeutics; programs; promoter; therapeutic gene; transduction efficiency; transgene expression; vector

Gene Vector and Production Core 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.

基因载体与生产核心 1.核心功能： 这个病毒载体的核心功能是为四个项目各提供两大服务： (1)设计和构建用于包装在病毒载体中的转基因； (2)病毒载体的生产、纯化和检测。 包装质粒的设计和构建包括： A)每个病毒载体都有特定的设计要求，包括包装能力、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)每个包装质粒的构建将通过限制性内切酶和测序来确认，以及 然后进行适当的转基因表达测试。 生产、提纯和测试包括： A)制作： AAV血清型1-9型 腺病毒2型 VSVG假型慢病毒 B)将使用氯化铯梯度离心法对AAV和腺病毒和蔗糖垫进行纯化 用于慢病毒生产的梯度，检查生产后的净化方案。 C)每种病毒的测试将包括： I)在交付给项目负责人之前，对透析后的病毒进行滴定。 二)检测病毒在HeLa细胞中对CMV或其他强毒的体外转导效率 启动子，或原代新生大鼠心肌细胞的限制性(心脏)启动子。 三)将使用Western印迹分析来确定病毒转基因在交付给 项目负责人。 该核心将制造和提纯AAV血清型1-9、腺病毒2型和慢病毒。具体来说， 我们将发展和维持大规模生产特定转基因的细胞系(100个15厘米的平板)。 由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、抑制和微RNA以及病毒的增强子-启动子配置 根据项目负责人的需要进行开发。核心D的主要功能是：1)生产腺苷 腺相关病毒，2)这些病毒的纯化，以及3)在转移到 科学计划的成员。另一个功能将是生成VSVG伪类型化 慢病毒，根据科学计划成员的需要。