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。内分泌破坏化学药品（EDCS）干扰了激素的复杂交通 控制人体中的每个器官和系统，并引起随之而来的发育和 具有重大人类健康关注的生殖作用。主要的筛选计划已经 在全球范围内识别和描述具有内分泌破坏者特征的化学物质的作用， 但是，由于需要表征复杂的EDC动作模式叠加的过程，因此该过程具有挑战性 与数百种循环激素相同的生物网络，它们发挥广泛 组织/器官，年龄和性别特异性效应。尽管这些复杂的全身EDC疾病表现，但 试图描述EDC活性的常规测定基于体外测定法，该测定法 孤立细胞培养物中的作用机理。这些测定无法揭示组织和生命阶段特异性 EDC的特性，很少提供有关关键毒理学终点的细节。相反，全体生物 体内测定被认为更适合获取此信息，斑马鱼被用作 这样做的主要模型。已经设计了转基因斑马鱼表达荧光报告蛋白 监视EDC暴露效果。但是，随着斑马鱼的年龄，它会累积荧光色素沉着 在其组织内部具有相应的光学清晰度丧失，使目标荧光信号歧视 在短短几天内实用，因此留下了太多的临床价值信息。 整合生物发光记者系统的斑马鱼转基因可能会解决此问题，因为斑马鱼 不要显示自然的生物发光，因此呈现出较高的信噪比。我们有 合成优化细菌荧光素酶（LUX）生物发光记者盒以有效表达 在真核基因对照中，在哺乳动物细胞和啮齿动物模型中证明了应用。我们 假设我们可以使用我们的密码子优化策略来设计一个可以作为细菌荧光素酶 在斑马鱼中有效表达，并与扩增的雌激素受体融合 方法，作为实时，组织特异性生物发光的新型体内转基因模型 EDC筛选在所有斑马鱼生活阶段。这项研究工作的具体目的是1）表达 在放大的雌激素受体融合下，斑马鱼中的密码子优化细菌荧光素酶，2）验证和 表征EDC暴露响应特征与一系列目标测试化合物的特征，3） 研究组织和生命阶段特异性的生物发光响应谱图对EDC暴露的全部暴露 斑马鱼发展的阶段。这项研究工作支持NIH NIEHS国家毒理学的愿景 计划“完善传统毒理学分析并开发基于机制的快速，基于机制的预测屏幕 环境引起的疾病”，并在旨在智力刺激的研究环境中这样做 并挑战四名本科生。