SITE-SPECIFIC RECOMBINATION TOOLS FOR C ELEGANS

C 线虫的位点特异性重组工具

• 批准号: 6074306
• 负责人: Owen Hughes
• 金额: $ 11.35万
• 依托单位: EON CORPORATION
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-05-01 至 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6074306
• 关键词: Caenorhabditis elegans; artificial chromosomes; expression cloning; gene targeting; genetic markers; genetic screening; genetic techniques; green fluorescent proteins; microinjections; polymerase chain reaction; recombinant DNA; recombinase; reporter genes; site directed mutagenesis; technology /technique development

C. elegans is a remarkably good system for studying issues of development and in many ways bridge the experimental and informational gap between single cell and complex animal models. To enable better understanding of gene function throughout development and within cell lineage's we aim to develop a system for lox/Cre recombination mediated control of gene expression. We will first test the ability of Cre recombinase to turn on a GFP reporter construct. lox sites will flank the GFP gene such that lox/Cre recombination will invert the gene and allow for its transcription. We also aim to develop targeted chromosomal integration via high efficiency site- specific recombination, which may eventually enable development of the first cDNA screening scale transformation technology in metazoans. We will test the ability of Cre recombination to direct transgene integration into a chromosomally placed lox site. This proposal holds two main points of innovation. First, efficient site specific recombination may bypass the requirement of introducing large quantities of DNA (for extrachromosomal array formation) and open up the potential of transformation by population scale technologies such as electroporation or coated particle bombardment. Second, the use of site specific recombination to directly integrate a transgene may bypass the mechanisms that shut down transgenes expression. PROPOSED COMMERCIAL APPLICATIONS: Eon intends to commercialize this lox/Cre work through: 1) sub-licensing arrangements within industry, 2) in-house contract research using expertise and materials developed with the lox/Cre system, and 3) in-house use of the lox/Cre transformation system for the identification of drug targets and the targets of drug tonicities. Sale of the system (lox containing worms and ox containing transformation vector) and supplies (injection quality Cre recombinase) in the form of a kit to academic researchers may be considered if no commercial source of injection quality Cre is found.

C. elegans是研究发育问题的一个非常好的系统，并且在许多方面弥合了单细胞和复杂动物模型之间的实验和信息差距。为了能够更好地了解基因功能的整个发展和细胞谱系内，我们的目标是开发一个系统lox/Cre重组介导的基因表达的控制。我们将首先测试Cre重组酶打开GFP报告构建体的能力。lox位点将位于GFP基因的侧翼，使得lox/Cre重组将反转基因并允许其转录。我们还旨在通过高效位点特异性重组开发靶向染色体整合，这可能最终能够在后生动物中开发第一个cDNA筛选规模的转化技术。我们将测试Cre重组指导转基因整合到染色体定位的lox位点的能力。该提案有两个主要创新点。首先，有效的位点特异性重组可以绕过引入大量DNA（用于染色体外阵列形成）的要求，并通过群体规模技术（如电穿孔或包被粒子轰击）打开转化的潜力。其次，使用位点特异性重组直接整合转基因可以绕过关闭转基因表达的机制。拟议的商业应用：Eon打算通过以下方式将该lox/Cre工作商业化：1）行业内的分许可安排，2）使用lox/Cre系统开发的专业知识和材料进行内部合同研究，以及3）内部使用lox/Cre转化系统识别药物靶标和药物张力靶标。如果没有发现注射质量Cre的商业来源，则可以考虑以试剂盒的形式向学术研究人员销售系统（含有蠕虫的lox和含有转化载体的牛）和供应品（注射质量Cre重组酶）。