Transgenic tools for Gal4 regulated gene expression in zebrafish

Gal4 调节斑马鱼基因表达的转基因工具

• 批准号: 7499962
• 负责人: MARNIE E HALPERN
• 金额: $ 33.24万
• 依托单位: CARNEGIE INSTITUTION OF WASHINGTON, D.C.
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7499962
• 关键词: Ablation; Address; Adult; Asses; Behavior; Binding; Biosensor; Brain; Bypass; Cell Death; Cells; Cellular Morphology; Cellular Structures; Chimeric Proteins; Cloning; Collaborations; Collection; Communities; Complex; Complication; DNA Sequence; Deposition; Development; Disease model; Drosophila genus; Effectiveness; Effector Cell; Embryo; Enhancers; Enzymes; Fishes; Gal-VP16; Galactose; Gene Expression; Gene Silencing; Gene Targeting; Generations; Genes; Genetic; Genetic Transcription; Germ Lines; Goals; Green Fluorescent Proteins; Invertebrates; Label; Laboratories; Lethal Genes; Maps; Mediating; Methodology; Methods; Methylation; Modeling; Monitor; Neurons; Numbers; Organ; Pathway interactions; Patient Self-Report; Physiological; Physiology; Plasmids; Process; Production; Proteins; Public Health; Reagent; Regulation; Reporter; Repression; Research; Research Personnel; Resources; Signal Pathway; Site; Staging; Synapses; System; Testing; Time; Tissues; Transcription Coactivator; Transcriptional Activation; Transgenes; Transgenic Organisms; Upper arm; Variant; Work; Yeasts; Zebrafish; base; cell type; design; fly; gene function; gene repression; improved; in vivo; insight; interest; promoter; protein function; research study; temperature sensitive mutant; tool; transcription factor; vector; zebrafish genome

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.

描述（由申请人提供）：大量在体内操纵基因作用的工具，主要通过使用来自酵母的双部转录调节系统，为无脊椎动物模型果蝇的研究带来了一个新的复杂水平。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。通过将这些上游激活序列（UAS）引入最小启动子和任何感兴趣的基因，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细胞或组织中在给定的时间被诱导。这种强大的方法不仅加强了发育研究和新疾病模型的产生，而且为神经元如何调节成人大脑中的复杂行为提供了见解。提出的工作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，产生了一种新的载体，通过转位整合整个斑马鱼基因组，从而将Gal4基因置于邻近组织特异性增强子的控制之下。此外，这些基因/增强子陷阱可以激活UAS控制下的其他基因，包括亚细胞结构的荧光报告基因和细胞死亡效应基因。许多研究人员已经要求构建质粒，并从这项工作中获得Gal4驱动基因和UAS报告基因的转基因系。虽然目前在斑马鱼中常规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。几种方法已经在果蝇中成功应用，并将测试它们在转基因斑马鱼中诱导Gal4活性的有效性。一些恢复的转基因系在UAS水平上表现出明显的转录沉默。本研究的另一个目的是利用这些插入来更好地了解UAS调控基因是如何沉默的，目的是设计新的转基因载体，使其不容易受到转录抑制。此外，一系列在无人机控制下的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方面具有广泛的用途。提出的工具集合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Gal4/UAS转基因系一样，将作为研究界的宝贵资源。