Development of gene targeting in C. elegans and D. rerio using zinc finger

描述(申请人提供)：这项研究的目标是开发两种重要的遗传模式生物-线虫秀丽线虫和斑马鱼Danio rerio的特定目标基因的精确修饰方法。线虫和鱼类都是将优雅的发育生物学与大规模遗传学相结合的强大的实验系统。这两个系统都有助于我们理解癌症生物学中的基本问题，包括细胞程序性死亡、器官发生的控制、癌症易感基因与环境的相互作用以及黑色素瘤的遗传学。这些模型系统的一个重要局限性是，目前还不能在线虫或FISH中获得对基因组进行定点操作的技术。因此，与小鼠胚胎干细胞和酵母不同，敲除特定基因或精确控制基因表达的时间和地点是不可能的。在过去的两年里，一种强大的基因靶向的新方法已经被开发出来，并成功地应用于苍蝇和哺乳动物的体细胞。这项技术使用嵌合锌指核酸酶来刺激特定基因在其天然基因组环境中的精确靶向。这项建议的目的是通过锌指核酸酶介导的同源重组在线虫和线虫中诱导有针对性的、可遗传的遗传变化。首先，我们将使用一种特性良好的锌指核酸酶来识别绿色荧光蛋白(GFP)基因。我们将把核酸酶引入到表达GFP的转基因线虫和斑马鱼中。我们预计由此产生的双链DNA断裂会刺激突变的非同源末端连接(NHEJ)，导致GFP信号的丢失。在第二阶段，我们将同时引入核酸酶和修复模板，这将使我们能够通过同源重组在目标基因座上创建精确的突变。在这项工作取得成功的基础上，我们将通过设计新的核酸酶并在体外和体内测试它们针对目标基因的活性，从而针对蠕虫和鱼类中的天然基因进行靶向。我们预计，如果成功，这种新的方法将是一种实用、灵活和强大的技术，将得到广泛的应用，显著增加这些系统对人类癌症生物学的照明能力。

DESCRIPTION (provided by applicant): The goal of this research is to develop methods for the precise modification of specific target genes in two important genetic model organisms, the nematode Caenorhabditis elegans and the zebrafish Danio rerio. Both nematodes and fish are powerful experimental systems that combine elegant developmental biology with large scale genetics. Both systems have contributed to our understanding of fundamental problems in cancer biology, including programmed cell death, the control of organogenesis, the interaction of cancer susceptibility genes with the environment, and the genetics of melanoma. An important limitation of these model systems is that techniques for site-specific manipulation of the genome are not currently available in either nematodes or fish. Thus, in contrast to murine embryonic stem cells and the yeast S. cerevisiae, it is not possible to knock out specific genes or to precisely control the time and place of gene expression. In the last two years, a powerful new approach to gene-targeting has been developed and successfully used in flies and in mammalian somatic cells. This technique uses chimeric zinc finger nucleases to stimulate precise targeting of specific genes in their native genomic context. The aim of this proposal is to induce targeted, heritable genetic changes via zinc finger nuclease-mediated homologous recombination in C. elegans and D. rerio. Initially we will employ a well-characterized zinc finger nuclease that recognizes the green fluorescent protein (GFP) gene. We will introduce the nuclease into transgenic nematodes and zebrafish that express GFP. We expect the resulting double-strand DNA breaks to stimulate mutagenic non-homologous end joining (NHEJ), leading to the loss of GFP signal. In the second phase, we will simultaneously introduce the nuclease and a repair template that will allow us to create precise mutations in the target locus by homologous recombination. Based on the success of this work we will then target native genes in the worm and the fish by designing novel nucleases and testing them in vitro and in vivo for activity against the targeted gene. We expect that, if successful, this novel approach would be a practical, flexible, and powerful technique that would find wide application, significantly increasing the power of these systems to illuminate human cancer biology.