Gene Targeting in Zebrafish

描述(申请人提供)：斑马鱼(Danio Rerio)是脊椎动物发育和人类疾病的杰出模型。近年来，该领域取得了巨大的发展，为我们理解人类发育缺陷的基因和机制做出了重大贡献。然而，使小鼠成为主要模型系统的两个最重要的工具，即定点突变和特定基因的定向插入，在斑马鱼中是缺乏的。这个项目的总体目标是通过开发斑马鱼基因打靶技术来纠正这个问题，该项目是一个拥有斑马鱼发育遗传学专业知识的实验室和一个拥有重组生物化学专业知识的实验室之间的合作。我们的方法是从果蝇的成功实验中改编而来，并基于被设计的称为锌指核酸酶(ZFN)的酶对选定的目标基因的切割。这项拟议的研究将在斑马鱼胚胎中表达ZFN，以产生目标基因的双链断裂，从而刺激局部重组。非同源末端连接引入序列变化，导致目标基因的小突变，从而允许斑马鱼的定向突变(基因敲除)。ZFN在工程供体分子存在的情况下诱导目标基因的双链断裂，导致同源重组，允许将任何所需序列精确地引入目标基因(基因敲入)。这项探索性的方案利用两个具有发育意义的基因中的三个靶序列来分析ZFN诱导斑马鱼突变的效用。我们将开发这项技术，目标是将靶向基因敲除和敲入技术的优势带到斑马鱼研究界。这些操纵斑马鱼基因组的新方法将使人们能够创建更好的人类发育缺陷和疾病模型。

DESCRIPTION (provided by applicant): Zebrafish (Danio rerio) is an outstanding model of vertebrate development and human disease. The field has grown enormously in recent years and made major contributions to our understanding of the genes and mechanisms underlying human developmental defects. However, two of the most important tools that made mice a premier model system, site-directed mutagenesis and targeted insertion of alterations into a specific gene, are lacking in zebrafish. The overall aim of this project, which is a collaboration between a lab with expertise in zebrafish developmental genetics and a lab with expertise in the biochemistry of recombination, is to rectify this problem by developing gene targeting technology for zebrafish. Our approach is adapted from successful experiments in Drosophila and is based on cleavage of the chosen target gene with designed enzymes called zinc finger nucleases (ZFNs). The proposed research will express ZFNs in zebrafish embryos to generate a double-strand break in a target gene, which stimulates local recombination. Non-homologous end joining introduces sequence changes that result in small mutations in the target gene, allowing targeted mutagenesis (gene knock-out) in zebrafish. ZFN-induced double-strand breaks in the target gene in the presence of engineered donor molecules leads to homologous recombination, allowing precise introduction of any desired sequence into the targeted gene (gene knock-in). This exploratory proposal uses three target sequences in two developmentally significant genes to analyze the utility of ZFN- induced mutagenesis in zebrafish. We will develop this technology with the goal of bringing the advantages of targeted gene knock-out and knock-in technology to the zebrafish research community. These novel approaches to manipulate the zebrafish genome will allow the creation of better models of a wide range of human developmental defects and diseases.