From luc to GFP to lux: Evolving an improved zebrafish model for the screening of

从 luc 到 GFP 再到 lux：改进斑马鱼模型以筛选

• 批准号: 8689387
• 负责人: STEVEN A RIPP
• 金额: $ 37.25万
• 依托单位: UNIVERSITY OF TENNESSEE KNOXVILLE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-15 至 2018-06-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8689387
• 关键词: Academic Research Enhancement Awards; Address; Affect; Age; Animals; Bacterial Luciferases; Binding; Biological Assay; Bioluminescence; Biosensing Techniques; Cell Culture Techniques; Characteristics; Chemical Actions; Chemical Exposure; Chemicals; Clinical; Codon Nucleotides; Complex; Data; Development; Developmental Process; Discrimination; Disease; Endocrine; Endocrine Disruptors; Endocrine disruption; Endocrine system; Engineering; Environment Design; Estrogen Receptors; Family; Fostering; Foundations; Genes; Genetic; Goals; Green Fluorescent Proteins; Growth; Health; Hormonal; Hormone Receptor; Hormones; Human; Human Cell Line; Human body; In Vitro; Left; Life; Link; Mammalian Cell; Metabolism; Modeling; Monitor; National Institute of Environmental Health Sciences; National Toxicology Program; Noise; Operon; Optics; Organ; Organism; Pigmentation physiologic function; Pigments; Problem Solving; Process; Property; Proteins; Reporter; Reporter Genes; Research; Rodent Model; Sensitivity and Specificity; Signal Transduction; Signaling Molecule; Spatial Distribution; Staging; System; Testing; Time; Tissues; Toxicology; Transgenic Model; Transgenic Organisms; United States National Institutes of Health; Vision; Whole Organism; Zebrafish; base; body system; chemical function; cofactor; design; environmental agent; high throughput screening; imaging modality; improved; in vitro Assay; in vivo; in vivo Model; next generation; programs; public health relevance; reproductive; response; screening; sex; tool; trafficking; undergraduate student; zebrafish development

Project Summary. Endocrine disrupting chemicals (EDCs) interfere with the intricate trafficway of hormones that control virtually every organ and system in the human body and elicit consequent developmental and reproductive effects that are of significant human health concern. Major screening programs have been established worldwide to identify and describe the actions of chemicals with endocrine disruptor characteristics, but the process is challenging because of the need to characterize complex EDC modes of action superimposed against an equally complex organismal network of hundreds of circulating hormones that exert widespread tissue/organ-, age-, and sex-specific effects. Despite these intricate whole-body EDC disease manifestations, conventional assays attempting to describe EDC activity are based on in vitro assays that profile chemical mechanisms of action in isolated cell cultures. These assays fail to reveal the tissue- and life stage-specific properties of EDCs and provide few details on critical toxicological endpoints. Conversely, whole-organism in vivo assays are considered more ideally suited for acquiring this information, with the zebrafish serving as a premier model for doing so. Transgenic zebrafish expressing fluorescent reporter proteins have been designed to monitor for EDC exposure effects. However, as the zebrafish ages it accumulates fluorescent pigmentation within its tissue with corresponding loss of optical clarity, making the discrimination of target fluorescent signals practical over only a few days and thus leaving behind far too much information of significant clinical value. Zebrafish transgenics that integrate bioluminescent reporter systems may solve this problem because zebrafish do not display natural bioluminescence and therefore present superior signal-to-noise ratios. We have synthetically optimized the bacterial luciferase (lux) bioluminescent reporter cassette for efficient expression under eukaryotic genetic controls with demonstrated application in mammalian cells and rodent models. We hypothesize that we can use our codon optimization strategy to design a bacterial luciferase that can as well be efficiently expressed in zebrafish and, in association with an amplified estrogen receptor fusion approach, applied as a new in vivo transgenic model for real-time, tissue-specific bioluminescent-based EDC screening across all zebrafish life stages. The specific aims of this research effort are to 1) Express a codon-optimized bacterial luciferase in zebrafish under an amplified estrogen receptor fusion, 2) Validate and characterize EDC exposure response characteristics against a battery of target test compounds, and 3) Investigate tissue- and life stage-specific bioluminescent response profiles to EDC exposures throughout all stages of zebrafish development. This research effort supports the vision of the NIH NIEHS National Toxicology Program to "refine traditional toxicology assays and develop rapid, mechanism-based predictive screens for environmentally induced diseases" and does so in a research environment designed to intellectually stimulate and challenge four undergraduate students.

项目摘要。内分泌干扰物（EDCs）干扰激素的复杂运输途径 它几乎控制着人体的每一个器官和系统， 对人类健康有重大影响的生殖影响。主要的筛查项目已经 在世界范围内建立，以确定和描述具有内分泌干扰物特性的化学品的作用， 但由于需要表征叠加的复杂EDC作用模式， 对抗一个同样复杂的生物网络，它由数百种循环激素组成， 组织/器官、年龄和性别特异性效应。尽管有这些复杂的全身EDC疾病表现， 试图描述EDC活性的常规测定是基于描述化学物质的体外测定 在分离的细胞培养物中的作用机制。这些分析未能揭示组织和生命阶段特异性 内分泌干扰物的性质，并提供了一些关于关键毒理学终点的细节。相反，整个有机体 体内测定被认为更理想地适合于获得这种信息，斑马鱼作为 第一个模型，这样做。已经设计了表达荧光报告蛋白的转基因斑马鱼， 监测EDC暴露效应。然而，随着斑马鱼年龄的增长， 在其组织内具有相应的光学清晰度损失， 仅在几天内实用，因此留下了太多具有重要临床价值的信息。 整合生物发光报告系统的斑马鱼转基因可能会解决这个问题，因为斑马鱼 不显示天然的生物发光，因此具有上级信噪比。我们有 合成优化的细菌荧光素酶（lux）生物发光报告盒用于有效表达 在哺乳动物细胞和啮齿动物模型中的应用得到证实。我们 假设我们可以使用我们的密码子优化策略来设计一种细菌荧光素酶， 在斑马鱼中有效表达，并与扩增的雌激素受体融合蛋白结合， 方法，作为一种新的体内转基因模型，用于实时，组织特异性生物发光为基础的 在所有斑马鱼生命阶段进行EDC筛查。本研究工作的具体目标是：1）表达一个 在扩增的雌激素受体融合下，斑马鱼中密码子优化的细菌荧光素酶，2） 表征针对一组目标测试化合物的EDC暴露响应特征，以及3） 研究组织和生命阶段特异性生物发光反应曲线，以EDC暴露在所有 斑马鱼的发育阶段这项研究工作支持了NIH NIEHS国家毒理学的愿景 该项目旨在“改进传统毒理学测定，并开发快速的、基于机制的预测性筛选， 环境引起的疾病”，并在一个旨在激发智力的研究环境中这样做， 挑战四名本科生