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，这一领域发展迅速，对我们理解人类发育缺陷的基因和机制作出了重大贡献。然而，使小鼠成为首要模型系统的两个最重要的工具，位点定向突变和靶向插入特定基因的改变，在斑马鱼中是缺乏的。该项目的总体目标是通过开发斑马鱼的基因靶向技术来解决这一问题，这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。我们的方法改编自果蝇的成功实验，并基于设计的酶锌指核酸酶（ZFNs）切割选定的靶基因。该研究将在斑马鱼胚胎中表达ZFNs，以在靶基因中产生双链断裂，从而刺激局部重组。非同源末端连接引入序列变化，导致靶基因发生小突变，从而在斑马鱼中实现靶向突变（基因敲除）。在工程供体分子存在的情况下，zfn诱导的靶基因双链断裂导致同源重组，允许将任何所需序列精确地引入靶基因（基因敲入）。本探索性建议利用两个重要发育基因中的三个靶序列来分析ZFN诱变在斑马鱼中的效用。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允许为广泛的人类发育缺陷和疾病创建更好的模型。

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.