Targeted Manipulation of the Zebrafish Genome through Homologous Recombination

描述（由申请人提供）：在许多方面，作为理解人类胚胎发育和疾病的模式生物，斑马鱼的效用超过了小鼠。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，目前缺乏的是针对斑马鱼基因组中特定（和临床相关）突变的同源重组技术（用于这些研究）。本提案的具体目的是通过采用已成功应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)作为转基因整合到基因组中的靶向载体，并在其两侧插入重组酶和核酸内切酶识别位点；2)一种可诱导的重组酶，将转基因作为一个封闭的环状染色体外载体从基因组中切除；3)在靶载体中产生DNA双链断裂（DSBs），使其重组的内切酶活性。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改变斑马鱼的基因组。为此，我们将开发以下技术组件和策略：A)靶向载体和选择。为了证明这一点，我们将首先以种系特异性基因vasa为目标。在初步研究中，我们设计了靶向载体，使其重组到vasa位点，从而特异性地驱动生殖细胞中GFP的表达。这将允许直接和有效的筛选遗传同源重组事件。B)靶向载体的解放：在第一种方法中，直接基于果蝇的方法，我们将利用Cre重组酶从基因组中去除靶向载体。然后，我们将尝试通过利用Tol2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，我们已经确定了两种在斑马鱼胚胎中起作用的大内切酶。我们建议利用这些酶在体内特异性地在染色体外靶向载体中产生dsb。D)重组时间：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表达内切酶和重组酶的活性，此时卵母细胞被引物进行同源重组。这将使基因靶向的效率最大化。将产生含有i)靶向载体，ii)重组酶和iii)核酸内切酶转基因元件的F0鱼。基因靶向事件将在卵巢中启动，并将通过筛选F1后代的生殖细胞特异性GFP表达来检测。

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.