Gene Targeting in Zebrafish

斑马鱼的基因靶向

• 批准号: 7026638
• 负责人: H. Joseph Yost
• 金额: $ 18.61万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-15 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7026638
• 关键词: active sites; animal breeding; animal genetic material tag; biotechnology; chimeric proteins; developmental genetics; embryo /fetus cell /tissue; embryogenesis; endoribonucleases; gene expression; gene mutation; gene targeting; genetically modified animals; green fluorescent proteins; microinjections; molecular genetics; nonmammalian vertebrate embryology; nuclease; protein structure; site directed mutagenesis; technology /technique development; zebrafish

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.

描述（由申请人提供）：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，该领域已经大大发展，并为我们对人类发育缺陷的基因和机制的理解做出了重大贡献。但是，在斑马鱼中缺乏两个使小鼠成为主要模型系统的最重要工具，即使小鼠成为主要的模型系统，定向诱变以及将改变的目标插入到特定基因中。该项目的总体目的是具有斑马鱼发育遗传学专业知识的实验室与具有重新组合生物化学专业知识的实验室之间的合作，是通过为斑马鱼开发基因靶向技术来纠正这一问题。我们的方法是根据果蝇中成功的实验改编而成的，并且是基于所选靶基因的裂解，该靶基因使用称为锌指核酸酶（ZFNS）的设计酶。拟议的研究将在斑马鱼胚胎中表达ZFN，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，导致靶基因中的突变，从而使斑马鱼中的靶向诱变（基因敲除）。在具有工程供体分子的存在下，ZFN诱导的靶基因中的双链断裂会导致同源重组，从而可以精确地将任何所需序列引入靶基因（Gene knock-In）。该探索性建议在两个发育意义的基因中使用三个目标序列来分析ZFN诱导的诱变在斑马鱼中的实用性。我们将开发这项技术，目的是将有针对性的基因敲除技术和敲除技术的优势带入斑马鱼研究界。这些操纵斑马鱼基因组的新型方法将允许创建各种人类发育缺陷和疾病的更好模型。