Transgenic tools for Gal4 regulated gene expression in zebrafish

描述(申请人提供)：大量在体内操纵基因作用的工具将无脊椎动物模型果蝇的研究带到了一个新的复杂水平，主要是通过使用从酵母改编的两部分转录调控系统。在这个系统中，转录因子Gal4与靶基因上游的特定DNA序列结合，激活其转录。通过在最小启动子和任何目的基因旁边引入这些上游激活序列(UA)，可以在Gal4存在的情况下获得高水平的表达。其他调节成分包括Gal80，一种与Gal4结合并限制其活性的抑制因子。通过在苍蝇的GAl4/UAS系统中建立时间和空间控制，任何基因都可以在给定的时间在给定的细胞或组织中诱导。这一强大的方法不仅加强了发育研究和新疾病模型的产生，还为神经元如何调节成人大脑的复杂行为提供了洞察力。拟议工作的目标是扩展和优化用于脊椎动物模型斑马鱼的多功能Gal4/UAS系统。在最初的研究中，产生了一种通过转座整合整个斑马鱼基因组的新载体，从而将Gal4基因置于相邻组织特异性增强子的控制之下。此外，这些基因/增强子陷阱还可以激活UAS控制下的其他基因，包括亚细胞结构的荧光报告程序和细胞死亡的效应因子。许多研究人员已经要求构建质粒和从这项工作中产生的Gal4驱动程序和UAS报告转基因系。虽然现在斑马鱼的基因表达通常是强健的、组织受限的，但还没有可靠的方法来暂时控制基因的表达。有几种方法已经在果蝇身上成功应用，并将测试它们在转基因斑马鱼中诱导Gal4活性的有效性。一些恢复的转基因株系在UAS水平上表现出明显的转录沉默。这项研究的另一个目的是利用这些插入来更好地了解UAS调控的基因是如何沉默的，目标是设计新的不太容易受到转录抑制的转基因载体。此外，UAS控制下的一系列新工具将在实时观察细胞形态、长期谱系研究和绘制大脑神经元连接图方面具有广泛的实用价值。拟议的工具集合将为斑马鱼实验带来新的和迫切需要的多功能性，并将成为研究界的宝贵资源，就像我们的第一组Gal4/UAS转基因株系一样。 与公共健康相关：利用酵母的Gal4/UAS转录激活系统在时间和空间上操纵基因表达的能力使果蝇模型的实验方法发生了革命性的变化。基于Gal4的通用方法允许研究人员使用荧光标记可视化细胞或亚细胞结构的子集，实时监测器官形成的基础过程，评估选择性组织或细胞中的蛋白质功能，并发现作用于遗传途径的新基因。将这一方法应用于斑马鱼具有无限的潜力，迫切需要解决早期发育以外的过程，如成年生理和行为。拟议的实验旨在继续三个研究小组之间富有成效的合作努力，以产生新的Gal4/UAS转基因工具，用于斑马鱼的调控基因表达。另一个目标是通过探索斑马鱼转基因转录沉默的基础和设计克服它的方法来优化Gal4/UAS系统。

DESCRIPTION (provided by applicant): A wealth of tools to manipulate gene action in vivo has brought a new level of sophistication to studies of the invertebrate model Drosophila, largely through the use of a bipartite transcriptional regulatory system adapted from yeast. In this system, the transcription factor Gal4 binds to specific DNA sequences upstream of target genes to activate their transcription. By introducing these upstream activating sequences (UAS) next to a minimal promoter and any gene of interest, high levels of expression are obtained in the presence of Gal4. Other regulatory components include Gal80, a repressor that binds to Gal4 and limits its activity. By building both temporal and spatial control into the GAl4/UAS system for the fly, any gene can be induced in a given cell or tissue at a given time. This powerful approach has not only enhanced developmental studies and the generation of new disease models, but has provided insights into how neurons mediate complex behaviors in the adult brain. The goal of the proposed work is to expand upon and optimize the versatile Gal4/UAS system for the vertebrate model, the zebrafish. In initial studies, a new vector was produced that integrates throughout the zebrafish genome by transposition, thereby placing the Gal4 gene under the control of adjacent tissue-specific enhancers. Moreover, these gene/enhancer traps could activate other genes under UAS control, including fluorescent reporters of sub-cellular structure and effectors of cell death. Numerous researchers have requested plasmid constructs and the Gal4 driver and UAS reporter transgenic lines produced from this work. While robust, tissue-restricted expression is routinely achieved now in zebrafish, there is no reliable method to control gene expression temporally. Several approaches have been successfully used in Drosophila and will be tested for their efficacy to induce Gal4 activity in transgenic zebrafish. Some recovered transgenic lines show pronounced transcriptional silencing at the level of the UAS. Another aim of this study is to take advantage of these insertions to gain a greater understanding of how UAS regulated genes are silenced, with the goal of designing new transgenic vectors less susceptible to transcriptional repression. In addition, a battery of new tools under UAS control will be produced that will have broad utility in observing cellular morphology real-time, in long-term lineage studies and in mapping neuronal connectivity in the brain. The proposed collection of tools will bring a new and much needed versatility to zebrafish experimentation and, as with our first set of Gal4/UAS transgenic lines, will serve as a valuable resource for the research community. PUBLIC HEALTH RELEVANCE: The ability to manipulate gene expression in time and space using the Gal4/UAS transcriptional activation system of yeast revolutionized experimental approaches in the Drosophila model. Versatile Gal4-based methods allow researchers to visualize subsets of cells or sub-cellular structures with fluorescent markers, to monitor processes that underlie organ formation real-time, to asses protein function in selective tissues or cells, and to discover new genes acting in genetic pathways. The application of this methodology to zebrafish has unlimited potential and is sorely needed to address processes beyond early development, such as adult physiology and behavior. The proposed experiments are aimed at continuing a productive collaborative effort between three research groups to generate new Gal4/UAS transgenic tools for regulated gene expression in the zebrafish. An additional goal is to optimize the Gal4/UAS system by exploring the basis of transcriptional silencing of zebrafish transgenes and devising approaches to overcome it.