SITE-SPECIFIC RECOMBINATION TOOLS FOR C ELEGANS

秀丽隐杆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差距。为了更好地了解基因在整个发育过程中的功能和细胞谱系内的功能，我们的目标是开发一个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报告基因结构的能力。lox位点位于GFP基因的侧面，这样lox/Cre重组将使基因反转并允许其转录。我们还致力于通过高效的位点特异性重组来开发靶向染色体整合，这可能最终使后生动物中第一个cDNA筛选规模转化技术的开发成为可能。我们将测试Cre重组引导转基因整合到染色体上放置的位点的能力。这个建议有两个主要的创新点。首先，高效的位点特异性重组可以绕过引入大量DNA（用于形成染色体外阵列）的要求，并通过群体规模技术（如电穿孔或包覆颗粒轰击）开辟转化的潜力。其次，使用位点特异性重组直接整合转基因可能会绕过关闭转基因表达的机制。拟议的商业应用：意昂打算通过以下方式将该lox/Cre工作商业化：1)行业内的子许可安排，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转化系统来识别药物靶标和药物毒性靶标。如果找不到商业上的注射级Cre来源，可以考虑以试剂盒形式向学术研究人员出售该系统（含蠕虫和含转化载体的lox）和用品（注射级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.