SITE-SPECIFIC RECOMBINATION TOOLS FOR C ELEGANS

线虫是研究发育问题的一个非常好的系统，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鸿沟。为了能够更好地了解基因在整个发育过程和细胞谱系中的功能，我们的目标是开发一种LOX/Cre重组介导的基因表达控制系统。我们将首先测试Cre重组酶开启GFP报告结构的能力。LOX位点将位于GFP基因的两侧，这样LOX/Cre重组将逆转该基因，并允许其转录。我们的目标也是通过高效的定点重组来开发有针对性的染色体整合，最终可能开发出第一个在后生动物中进行筛选的规模转化技术。我们将测试Cre重组将转基因整合到染色体上的lox位点的能力。这项提议有两个主要创新点。首先，高效的位点特异性重组可能绕过引入大量DNA(用于染色体外阵列形成)的要求，并打开群体规模技术(如电穿孔或包被粒子轰击)转化的潜力。其次，使用位点特异性重组来直接整合转基因可能绕过关闭转基因表达的机制。拟议的商业应用：意昂集团打算通过以下方式将这种LOX/CRE工作商业化：1)行业内的次级许可安排，2)内部合同研究，使用LOX/CRE系统开发的专业知识和材料，以及3)内部使用LOX/CRE转换系统来识别药物靶标和药物强度靶标。如果找不到商业来源的注射质量Cre，可以考虑以试剂盒的形式向学术研究人员出售该系统(含有蠕虫的LOX和含有转化载体的OX)和供应品(注射质量的Cre重组酶)。

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.