Salmonella-to-Eukaryotic Cell Multi-task Gene Delivery

沙门氏菌到真核细胞的多任务基因传递

• 批准号: 7054755
• 负责人: Donald G. Guiney
• 金额: $ 7.54万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7054755
• 关键词: Salmonella; biotechnology; eukaryote; gene delivery system; host organism interaction; microorganism conjugation; transfection; transfection /expression vector

DESCRIPTION (provided by applicant): The aims of this R03 proposal are to perform the initial experiments: 1. to develop a system of gene transfer from Salmonella to eukaryotic host cells; 2. to examine the Salmonella properties and host cell factors that affect gene transfer processes; and 3. compare conjugation with other Salmonella-based DNA transfer systems. The first aim emerges from our recent discovery of E. coli-to-mammalian cell conjugative DNA transfer. The methodology developed for the E. coli system will be adapted to donor strains of Salmonella, in order to ultimately capitalize on bacterial-host cell interactions that are unique to the Salmonella organism. With the noninvasive E. coli K12 donors, non-conjugative mechanisms of DNA transfer such as transformation and bacterial engulfment were conclusively ruled out by rigorous controls, including the use of Dnasel in the milieu and inhibitors of endocytosis. With the Salmonella system, the hypothesis is that more than one DNA delivery mechanism will be operative, since Salmonella has multiple ways of interacting with host cells. Such delivery mechanisms could involve intracellular ("intracytoplasmic") bacterial delivery, extracellular bacterial delivery, and intravacuole bacterial delivery. In each cellular locale, distinct processes can be at work. The second aim will help define the bacterial and host cell properties that impact these events. This approach will characterize the transfer mechanisms while providing ways to improve DNA delivery as a therapeutic tool. The third aim will use those defining factors to distinguish bacterial conjugative transfer by Salmonella from that of E. coli and to compare conjugative transfer with the other Salmonella-based DNA transfer systems. These studies are intended to develop attenuated Salmonella strains for DNA delivery for DNA vaccine approaches, corrective gene therapy, immunotherapy, and the prevention and treatment of certain kinds of cancer.

描述（由申请人提供）：本R 03提案的目的是进行初始实验：1.建立从沙门氏菌到真核宿主细胞的基因转移系统; 2.研究沙门氏菌的特性和影响基因转移过程的宿主细胞因素;以及3.比较与其他沙门氏菌为基础的DNA转移系统的结合。第一个目标来自我们最近发现的E.大肠杆菌到哺乳动物细胞的接合DNA转移。本文介绍了E.大肠杆菌系统将适应沙门氏菌的供体菌株，以最终利用沙门氏菌生物体特有的细菌-宿主细胞相互作用。非侵入性E. coli K12供体，通过严格的控制，包括在环境中使用Dnasel和内吞作用抑制剂，最终排除了DNA转移的非接合机制，如转化和细菌吞噬。对于沙门氏菌系统，假设不止一种DNA递送机制将起作用，因为沙门氏菌具有多种与宿主细胞相互作用的方式。这种递送机制可能涉及细胞内（“胞质内”）细菌递送、细胞外细菌递送和液泡内细菌递送。在每一个细胞区域，不同的过程可以在工作。第二个目标将有助于确定影响这些事件的细菌和宿主细胞特性。这种方法将表征转移机制，同时提供改善DNA递送作为治疗工具的方法。第三个目标是利用这些决定因素来区分沙门氏菌和大肠杆菌的接合转移。并与其他沙门氏菌DNA转移系统进行比较。这些研究旨在开发减毒沙门氏菌菌株，用于DNA疫苗方法，纠正基因治疗，免疫治疗以及预防和治疗某些类型的癌症。