Custom integration tools for functional genomics

功能基因组学的定制集成工具

• 批准号: 6646443
• 负责人: MICHELE P CALOS
• 金额: $ 15.36万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-30 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6646443
• 关键词: Escherichia coli; animal genetic material tag; biotechnology; functional /structural genomics; genetic manipulation; genetic screening; genetic techniques; genetically modified animals; intermolecular interaction; molecular shape; nucleic acid sequence; polymerase chain reaction; reagent /indicator; recombinase; robotics; site directed mutagenesis; technology /technique development

DESCRIPTION (provided by applicant): This proposal develops powerful new methods for obtaining efficient site-specific integration at pre-determined genomic sequences. The work elaborates on a successful line of research in my laboratory involving recombination mediated by phage integrases at their compact (30 - 40-bp) attB and attP recognition sites. We have demonstrated that this reaction represents the most efficient site-specific integration system developed to date for higher cells. In the proposed strategy, integration takes place between an attB site present on an incoming DNA vector and a naturally-occurring attP-like sequence located in the mouse genome. The result is covalent integration of the incoming DNA into a pre-specified position in the mouse genome. Directed evolution of phage integrases is used to produce a large library of integration tools of unprecedented efficiency and specificity. With this system, a custom integration tool can be designed for any genomic sequence and used to integrate a DNA insert of large or small size at high efficiency. The DNA to be inserted can be custom designed as desired. Because the method is site-specific, the location of the insertion is automatically known. We develop the appropriate DNA shuffling and genetic screening methods here for generation of a library of custom integration tools. The strategy is scalable to reach essentially all genes in the mouse genome.

描述（由申请人提供）：该提案开发了强大的新功能 在预定位置获得有效位点特异性整合的方法 基因组序列。这部作品详细阐述了我的一系列成功的研究 实验室涉及噬菌体整合酶介导的重组 紧凑 (30 - 40-bp) attB 和 attP 识别位点。我们已经证明了 该反应代表了最有效的位点特异性整合系统 迄今为止已开发用于高等细胞。在拟议的战略中，整合需要 位于传入 DNA 载体上存在的 attB 位点和 位于小鼠基因组中的天然存在的 attP 样序列。结果 是将传入的 DNA 共价整合到预先指定的位置 小鼠基因组。噬菌体整合酶的定向进化用于产生 具有前所未有的效率和特异性的大型集成工具库。 通过该系统，可以为任何基因组设计定制集成工具 序列并用于高整合大或小尺寸的DNA插入物 效率。待插入的 DNA 可以根据需要进行定制设计。因为 该方法是特定于站点的，插入的位置是自动的 已知。我们开发适当的 DNA 改组和基因筛选方法 此处用于生成自定义集成工具库。策略是 可扩展以触及小鼠基因组中的基本上所有基因。