Transgenic tools for Gal4 regulated gene expression in zebrafish

描述（由申请人提供）：大量的体内操纵基因作用的工具为无脊椎动物模型果蝇的研究带来了新的复杂程度，主要是通过使用从酵母中改造而来的二分转录调控系统。在该系统中，转录因子Gal 4与靶基因上游的特定DNA序列结合以激活其转录。通过在最小启动子和任何感兴趣的基因旁边引入这些上游激活序列（UAS），在Gal 4存在下获得高水平的表达。其他调节成分包括Gal 80，一种结合Gal 4并限制其活性的阻遏物。通过在果蝇的GA 14/UAS系统中建立时间和空间控制，可以在给定的时间在给定的细胞或组织中诱导任何基因。这种强大的方法不仅增强了发育研究和新疾病模型的产生，而且为神经元如何介导成人大脑中的复杂行为提供了见解。所提出的工作的目标是扩大和优化的通用Gal 4/UAS系统的脊椎动物模型，斑马鱼。在最初的研究中，产生了一种新的载体，通过转座整合整个斑马鱼基因组，从而将Gal 4基因置于相邻组织特异性增强子的控制下。此外，这些基因/增强子陷阱可以在UAS控制下激活其他基因，包括亚细胞结构的荧光报告基因和细胞死亡效应基因。许多研究人员已经要求质粒构建体和Gal 4驱动和UAS报告基因转基因系从这项工作中产生。虽然现在在斑马鱼中常规地实现了稳健的组织限制性表达，但没有可靠的方法来暂时控制基因表达。几种方法已成功地用于果蝇，并将测试其在转基因斑马鱼中诱导Gal 4活性的功效。一些恢复的转基因株系在UAS水平上显示出明显的转录沉默。本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，目的是设计新的转基因载体，使其不易受到转录抑制的影响。此外，将在UAS控制下生产一系列新工具，这些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方面具有广泛的实用性。所提出的工具集将为斑马鱼实验带来新的和急需的多功能性，并且与我们的第一组Gal 4/UAS转基因系一样，将作为研究界的宝贵资源。 公共卫生相关性：使用酵母的Gal 4/UAS转录激活系统在时间和空间上操纵基因表达的能力彻底改变了果蝇模型中的实验方法。基于Gal 4的多功能方法使研究人员能够用荧光标记物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，并发现在遗传途径中起作用的新基因。将这种方法应用于斑马鱼具有无限的潜力，并且迫切需要解决早期发育以外的过程，例如成年生理学和行为。拟议的实验旨在继续三个研究小组之间的富有成效的合作努力，以产生新的Gal 4/UAS转基因工具，在斑马鱼中调节基因表达。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Gal 4/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.