Targeted Manipulation of the Zebrafish Genome through Homologous Recombination

通过同源重组对斑马鱼基因组进行靶向操作

• 批准号: 7496679
• 负责人: Brian Ciruna
• 金额: $ 21.08万
• 依托单位: HOSPITAL FOR SICK CHLDRN (TORONTO)
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-18 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7496679
• 关键词: Alleles; Animal Model; Applications Grants; Behavior; Biological Assay; Biological Models; Cell Culture Techniques; Cell Lineage; Chimeric Proteins; Code; Congenital Abnormality; DNA; DNA Double Strand Break; Detection; Development; Disease; Drosophila genome; Drosophila genus; Elements; Embryo; Embryonic Development; Endonuclease I; Enzymes; Event; Fishes; Gene Targeting; Gene Transfer Techniques; Generations; Genes; Genetic; Genetic Markers; Genetic Recombination; Genetic Research; Genetic Screening; Genome; Germ Cells; Germ Lines; Green Fluorescent Proteins; Heat-Shock Response; Human; Image; Laboratories; Larva; Life; Mediating; Medical; Meiosis; Methodology; Methods; Modeling; Molecular Genetics; Mus; Mutate; Mutation; Nuclear; Oocytes; Optics; Ovary; Phenotype; Prophase; Public Health; Recombinant DNA; Regulation; Reporter; Reporting; Research Personnel; Resources; Screening procedure; Sequence Homologs; Site; Staging; Structure of primordial sex cell; Techniques; Technology; Therapeutic Agents; Time; Tissues; Transgenes; Transgenic Organisms; Transposase; Upper arm; Zebrafish; base; bioimaging; chemical addition; clinically relevant; design; embryonic stem cell; endodeoxyribonuclease SceI; endonuclease; gene function; homologous recombination; human disease; in vivo; insight; mutant; novel therapeutics; positional cloning; promoter; recombinase; small molecule; vector; vector genome; zebrafish genome

DESCRIPTION (provided by applicant): In many respects, the zebrafish surpasses the mouse in utility as a model organism for understanding human embryonic development and disease. Zebrafish embryos are optically clear and easily manipulated, permitting sub-cellular biomedical imaging studies in living tissue. Moreover, small molecule compounds can be easily applied to zebrafish larvae, allowing screens for novel therapeutic agents that are capable of suppressing mutant phenotypes. What is lacking, however, are homologous recombination technologies for targeting specific (and clinically relevant) mutations into the zebrafish genome (for use in these studies). The specific aim of this proposal is to develop gene-targeting technology for zebrafish, by adapting an in vivo transgene-based homologous recombination (HR) strategy that has been successfully used in Drosophila. The strategy involves three components: 1) a targeting vector that is integrated into the genome as a transgene, and flanked by recombinase and endonuclease recognition sites; 2) an inducible recombinase enzyme that will excise the transgene from the genome as a closed circular extra- chromosomal vector; and 3) an endonuclease activity that will generate DNA double-strand breaks (DSBs) in the targeting vector, rendering it recombinogenic. In Drosophila studies, this nuclear DNA fragment can efficiently recombine with homologous loci. We hypothesize that a similar approach can be used to mutate the zebrafish genome. To this end, we will develop the following technological components and strategies: A) Targeting vector and selection. As a proof of principal, we will first target vasa, a germ-line specific gene. In preliminary studies, we have designed the targeting vector such that recombination into the vasa locus will specifically drive GFP expression in germ cells. This will allow direct and efficient screening for heritable homologous recombination events. B) Liberation of targeting vector: In a first approach, directly based on the Drosophila methodology, we will utilize Cre recombinase to excise targeting vectors from the genome. We will then attempt to streamline the gene targeting strategy by utilizing Tol2 transposase activity to mobilize the vasa-GFP targeting vector. C) Generation of DSBs: In preliminary studies we have identified two mega-endonucleases that are functional in zebrafish embryos. We propose to utilize these enzymes to specifically generate DSBs in extra-chromosomal targeting vectors, in vivo. D) Timing of recombination: We will utilize an oocyte-specific promoter element to express endonuclease and recombinase activities during meiotic prophase 1, when the oocyte is primed for homologous recombination. This will maximize the efficiency of gene targeting. F0 fish that harbor i) targeting vector, ii) recombinase, and iii) endonuclease transgenic elements will be generated. Gene targeting events will initiate in the ovary, and will be detected by screening F1 progeny for germ cell-specific GFP expression. PUBLIC HEALTH RELEVANCE: Homologous recombination technologies, as developed in this proposal, will allow researchers to specifically mutate and manipulate the zebrafish genome. This will allow geneticists to I) target clinically relevant mutations into zebrafish, to mimic human birth defects and disease); and 2) generate "conditional" zebrafish mutants so that gene function can be studied in specific tissues or cell lineages, or at different stages of development. Using these new resources, researchers will be able to exploit the experimental advantages of zebrafish to gain new insights into the molecular and genetic mechanisms that control embryonic development, behavior and disease, and to discover novel therapeutic agents that can suppress mutant phenotypes and treat human disease.

描述（由申请人提供）：在许多方面，斑马鱼在作为了解人类胚胎发育和疾病的模式生物的实用性方面超过了小鼠。斑马鱼胚胎光学清晰，易于操作，允许在活组织中进行亚细胞生物医学成像研究。此外，小分子化合物可以很容易地应用于斑马鱼幼虫，允许筛选能够抑制突变表型的新型治疗剂。然而，缺乏的是同源重组技术，用于将特定的（和临床相关的）突变靶向到斑马鱼基因组中（用于这些研究）。该提案的具体目的是通过适应已成功用于果蝇的体内转基因同源重组（HR）策略，开发斑马鱼的基因靶向技术。该策略包括三个组成部分：1）靶向载体，其作为转基因整合到基因组中，并且侧翼为重组酶和核酸内切酶识别位点; 2）诱导型重组酶，其将从基因组切除转基因作为闭合的环状染色体外载体;和3）将在靶向载体中产生DNA双链断裂（DSB），使其成为重组原性的内切核酸酶活性。在果蝇研究中，该核DNA片段可以有效地与同源位点重组。我们假设类似的方法可以用来突变斑马鱼基因组。为此，我们将开发以下技术组件和策略：A）靶向载体和选择。作为原理的证明，我们将首先针对vasa，一个生殖系特异性基因。在初步研究中，我们已经设计了靶向载体，使得重组到vasa位点中将特异性地驱动GFP在生殖细胞中的表达。这将允许直接和有效地筛选可遗传的同源重组事件。B）靶向载体的释放：在第一种方法中，直接基于果蝇方法，我们将利用Cre重组酶从基因组切除靶向载体。然后，我们将尝试通过利用Tol 2转座酶活性来动员vasa-GFP靶向载体来简化基因靶向策略。C）DSB的产生：在初步研究中，我们已经鉴定了在斑马鱼胚胎中有功能的两种大内切核酸酶。我们建议利用这些酶在体内在染色体外靶向载体中特异性地产生DSB。D）重组的时间：我们将利用卵母细胞特异性启动子元件在减数分裂前期1期间表达核酸内切酶和重组酶活性，此时卵母细胞被引发进行同源重组。这将最大限度地提高基因打靶的效率。将产生携带i）靶向载体、ii）重组酶和iii）内切核酸酶转基因元件的FO鱼。基因靶向事件将在卵巢中启动，并将通过筛选F1子代的生殖细胞特异性GFP表达来检测。 公共卫生相关性：这项提案中开发的同源重组技术将使研究人员能够特异性地突变和操纵斑马鱼基因组。这将允许遗传学家I）将临床相关突变靶向斑马鱼，以模拟人类出生缺陷和疾病;和2）产生“条件性”斑马鱼突变体，以便可以在特定组织或细胞谱系中或在不同发育阶段研究基因功能。利用这些新资源，研究人员将能够利用斑马鱼的实验优势，对控制胚胎发育，行为和疾病的分子和遗传机制获得新的见解，并发现可以抑制突变表型和治疗人类疾病的新型治疗药物。