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

描述（由申请人提供）：内分泌干扰化学物质（EDCs）干扰控制人体几乎每个器官和系统的激素的复杂传输方式，并引起随之而来的对人类健康有重大影响的发育和生殖影响。世界各地已经建立了主要的筛选项目，以识别和描述具有内分泌干扰物特征的化学物质的作用，但这一过程具有挑战性，因为需要表征复杂的EDC作用模式，这些模式叠加在数百种循环激素组成的同样复杂的有机体网络上，这些激素发挥着广泛的组织/器官、年龄和性别特异性作用。尽管有这些复杂的全身EDC疾病表现，但试图描述EDC活性的传统检测方法是基于在体外分析分离细胞培养物中作用的化学机制。这些检测无法揭示EDCs的组织和生命阶段特异性特性，也无法提供关键毒理学终点的详细信息。相反，整个生物体的体内分析被认为更适合于获得这些信息，斑马鱼是这样做的首要模型。表达荧光报告蛋白的转基因斑马鱼被设计用于监测EDC暴露效应。然而，随着斑马鱼年龄的增长，斑马鱼在其组织内积累了荧光色素沉着，光学清晰度随之下降，使得目标荧光信号的识别仅在几天内就能实现，从而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以解决这一问题，因为斑马鱼不表现出天然的生物发光，因此具有优越的信噪比。我们对细菌荧光素酶（lux）生物发光报告盒进行了综合优化，使其在真核遗传控制下有效表达，并在哺乳动物细胞和啮齿动物模型中得到了应用。我们假设我们可以使用我们的密码子优化策略来设计一种细菌荧光素酶，这种荧光素酶可以在斑马鱼中有效表达，并与放大的雌激素受体融合方法相结合，作为一种新的体内转基因模型，用于在斑马鱼的所有生命阶段进行实时的、组织特异性的基于生物发光的EDC筛选。本研究的具体目的是：1)在放大的雌激素受体融合下，在斑马鱼中表达密码子优化的细菌荧光素酶；2)验证和表征针对一系列目标测试化合物的EDC暴露反应特征；3)研究斑马鱼发育各个阶段对EDC暴露的组织和生命阶段特异性生物发光反应谱。这项研究工作支持NIH NIEHS国家毒理学计划的愿景，即“改进传统的毒理学分析，开发快速的、基于机制的环境诱发疾病的预测筛选”，并在一个旨在刺激和挑战四名本科生的研究环境中完成。

DESCRIPTION (provided by applicant): Endocrine disrupting chemicals (EDCs) interfere with the intricate traffic way 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 efficiet 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.