CORE--GENE TRANSFER

近年来，基因转移技术无论是在病毒载体的辅助下还是以裸DNA的形式被广泛应用于阐明调节血管功能和血压的机制。项目调查员在核心主任的帮助下产生的初步数据表明，这项技术为方案项目的每个组成部分提供了有用的工具。核心主任与项目研究人员合作了十多年，最近开发和交付了以CYP4A和HO基因为主的重组逆转录病毒表达载体和病毒(腺病毒和逆转录病毒)，用于项目研究人员提出的体外和体内研究。该核心将执行以下功能：1)使用带有和不带有GFP或β-半乳糖标签的商用载体构建重组的CYP4A、HO和COX-2腺病毒和逆转录病毒；2)使用带有和不带有GRP或β-半乳糖标签的商用载体构建腺病毒和逆转录病毒；2)分离、纯化和繁殖重组病毒载体；3)确定重组病毒载体的滴度并保持库存；4)维持各种质粒和有能力的包装细胞的库存；5)开发携带组织和细胞系特异性启动子的载体；以及6)为每个研究人员提供评估转基因表达的资源和专业知识，并与项目研究人员合作，使用RT/PCR、Southern印迹分析和GFP或β-Gal的检测来验证转基因在体外和体内的表达。在分子试剂的产生和生产、实验设计和数据解释方面，项目调查人员将得到核心主任和技术人员的支持。

Gene transfer technology either with the aid of viral vectors or in the form of naked DNA has been used extensively in recent years to elucidate mechanisms regulating vascular function and blood pressure. Preliminary data generated by project investigators with the help of the Core director indicate that this technology provides useful tools to each of the program project components. The core director collaborated with project investigators for more than ten years and recently produced and delivered recombinant retroviruses expression vectors and viruses (adenoviruses and retroviruses) containing primarily the CYP4A and HO genes for in vitro and in vivo studies proposed by the project investigators. The core will perform the following functions; 1) construct recombinant CYP4A, HO and COX-2 adenoviruses and retroviruses using commercially available vectors with and without GFP or beta-gal tagging; 2) adenoviruses and retroviruses using commercially available vectors with and without GRP or beta-gal tagging; 2) isolate and purify and propagate recombinant viral vectors; 3) determine titers and maintains stocks of recombinant viral vectors; 4) maintain stocks of the various plasmids and competent and packaging cells; 5) develop vectors carrying tissue and cell lineage specific promoters; and 6) provide the resources and expertise to each investigator for evaluating transgene expression and work along with the project investigators in validating transgene expression in vitro and in vivo using RT/PCR, Southern blot analysis and detection of GFP or beta-gal. Project investigators will be supported by the core director and the technical staff in the generation and production of molecular reagents, experimental design and data interpretation.