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）：在体内操纵基因作用的大量工具为无脊椎动物模型果蝇的研究带来了新的精致水平，主要是通过使用由酵母改编的双分部分的转录调节系统。在该系统中，转录因子GAL4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引入这些上游激活序列（UAS），可以在GAL4存在下获得高水平的表达。其他调节组件包括Gal80，Gal80是一种与GAL4结合并限制其活性的阻遏物。通过将时间和空间控制构建到GAL4/UAS系统的苍蝇中，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展研究和新疾病模型的产生，而且还提供了有关神经元如何介导成人大脑复杂行为的见解。提议的工作的目的是扩展并优化脊椎动物模型斑马鱼的多功能GAL4/UAS系统。在最初的研究中，产生了一个新的载体，该载体通过转座整合整个斑马鱼基因组，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/增强子陷阱可以在UAS控制下激活其他基因，包括细胞生死亡的荧光记者和细胞死亡的效应因子。许多研究人员都要求质粒构建体以及这项工作产生的GAL4驱动器和UAS记者转基因系。虽然在斑马鱼中常规实现组织限制的表达，但没有可靠的方法可以在时间上控制基因表达。已在果蝇中成功使用了几种方法，并将测试其在转基因斑马鱼中诱导GAL4活性的功效。一些回收的转基因线在UAS的水平上显示出明显的转录沉默。这项研究的另一个目的是利用这些插入，以对UAS受调节的基因的沉默有更深入的了解，其目的是设计新的转基因矢量不容易受到转录抑制的影响。此外，将产生一系列新工具，这些工具将在观察实时观察细胞形态，长期谱系研究和映射大脑中的神经元连接方面具有广泛的实用性。拟议中的工具集合将为斑马鱼实验带来新的急需多功能性，并且与我们的第一组GAL4/UAS转基因线一样，它将成为研究社区的宝贵资源。 公共卫生相关性：使用酵母模型中酵母的GAL4/UAS转录激活系统操纵基因表达的能力。基于多功能GAL4的方法使研究人员能够可视化细胞或带有荧光标记的细胞下结构的子集，以监测实时器官形成基础的过程，从而使蛋白质在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。这种方法在斑马鱼中的应用具有无限的潜力，并且需要解决早期发展以外的过程，例如成人生理和行为。所提出的实验旨在继续在三个研究小组之间继续进行富有成效的协作努力，以生成新的GAL4/UAS转基因工具，用于调节斑马鱼的基因表达。另一个目标是通过探索斑马鱼转基因的转录沉默和设计方法来克服它来优化GAL4/UAS系统。