Design of a high-throughput screen for chemicals that cause meiotic aneuploidy

导致减数分裂非整倍体化学物质的高通量筛选设计

• 批准号: 8586524
• 负责人: Patrick Allard
• 金额: $ 24.67万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-12-01 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8586524
• 关键词: Affect; Aneugens; Aneuploidy; Animal Model; Animals; Anti-Anxiety Agents; Biochemical; Biochemical Process; Biological; Biological Assay; Biological Models; Birth; Caenorhabditis elegans; Cell Culture Techniques; Cell division; Cell physiology; Cells; Chemicals; Chromosome Segregation; Chromosome abnormality; Chromosomes; Collaborations; Complex; Congenital Abnormality; Data; Databases; Defect; Detection; Development; Embryo; Environment; Environmental Exposure; Environmental Impact; Etiology; Event; Exposure to; Future; Generations; Genes; Genetic; Germ Cells; Germ cell tumor; Goals; Gonadal structure; Grant; Health; Human; In Vitro; Infertility; Lead; Libraries; Luciferases; Malignant Neoplasms; Mammals; Meiosis; Mitosis; Modeling; Molecular Analysis; Molecular Genetics; National Institute of Environmental Health Sciences; Nematoda; Organism; Outcome; Pathway interactions; Penetration; Pesticides; Phase; Plastics; Process; Production; Reporter; Research Personnel; Rodent Model; Spontaneous abortion; System; Technology; Testing; Time; Toxic Environmental Substances; Toxic effect; Training; Transgenic Organisms; Translations; Validation; Xenobiotics; base; bisphenol A; chemotherapeutic agent; design; environmental chemical; gene conservation; high throughput screening; improved; in vivo; insight; mutant; novel; prenatal; programs; reproductive; screening; stem; success; tool; toxicant; toxicant screening; trend

Aneuploidies, or abnormal numbers of chromosomes in a cell, originate from errors in segregation of chromosomes during the two highly regulated programs of cell division, mitosis and meiosis. The consequences of aneuploidies of meiotic origins are devastating as they contribute to 35% of clinically recognized miscarriages, 4% of still births, 20% of all birth defects and to virtually all germ cell tumors. There is also a clear environmental contribution in the etiology of meiotic chromosome segregation errors as compounds as diverse as plastics, pesticides and anti-anxiety drugs have been shown to cause meiotic aneuploidies in mammals. Despite the severe health outcomes of meiotic aneuploidies, the screening of environmental toxicants for their ability to disrupt meiosis and cause aneuploidy has been severely lagging. This problem is inherent to the complexity of the meiotic program which differs in part significantly from mitosis, cannot be efficiently recapitulated in vitro and initiates early on during mammalian development within the confines of the embryonic gonad. Thus, we currently lack the tools to efficiently and comprehensively interrogate our chemical environment for its effect on meiotically-derived aneuploidies. In this application, we propose the development a high-throughput screening approach that makes use of the remarkable biological features of the nematode Caenorhabditis elegans, which as a meiotic model system shows a high degree of conservation with humans. To this aim, we are building on a strategy developed for the isolation of mutants defective in chromosome segregation during meiosis. In the K99 section of the project, we will complete the changes in the design of the C. elegans transgenic strain used in this strategy to make it suitable for toxicant screening. These changes include a multi-assayable luciferase/GFP reporter system and an increased sensitivity by improving chemical penetration. We will test the validity of our approach by performing two small scale screens against compounds with known aneugenic activity and reproductive effects: one set of chemotherapeutic agents from the NCI and one of environmental toxicants from the EPA. In the second phase (R00) of the project, we will move our system to a large scale, high- throughput setting to screen two libraries, first the Toxcast Phase I 309 chemicals and then the Tox21 library of about 10,000 compounds. Secondary validation first in C. elegans and in mammalian models will conclusively establish the validity of the screen and likely lead to the identification of novel meiotic aneugenic compounds. The collaborative basis of this project which combines our expertise with that of key researchers at EPA and NIEHS and the tremendous training environment make us confident of the success of the approach. Together, we will be able, for the first time, to assess the meiotic aneugenic activity of our environment and improve our ability to predict toxicity to mammals and humans.

非整倍体，或细胞中异常数目的染色体，起源于错误的分离 染色体在细胞分裂、有丝分裂和减数分裂这两个高度调控的过程中。这个 起源于减数分裂的非整倍体的后果是毁灭性的，因为它们在临床上占35% 公认的流产，死产的4%，所有出生缺陷的20%，以及几乎所有生殖细胞肿瘤。的确有 在减数分裂染色体分离错误的病因学中也有明显的环境贡献，如 各种化合物，如塑料、杀虫剂和抗焦虑药物，都被证明会导致减数分裂。 哺乳动物中的非整倍体。 尽管减数分裂非整倍体带来了严重的健康后果，但环境毒物的筛选 因为它们破坏减数分裂并导致非整倍体的能力一直严重滞后。这个问题是固有的 减数分裂程序的复杂性在一定程度上不同于有丝分裂，不能有效地 在体外概括，并在哺乳动物发育早期胚胎的范围内启动 性腺。因此，我们目前缺乏有效和全面地审问我们的化学品的工具。 它对减数分裂衍生的非整倍体的影响的环境。 在这个应用中，我们提出了开发一种高通量的筛选方法，该方法利用 秀丽线虫作为减数分裂模式的显著生物学特征 系统显示出与人类高度保守的关系。为此，我们正在制定一项战略 用于分离减数分裂过程中染色体分离缺陷的突变体。在K99区 在该项目中，我们将完成本项目中使用的线虫转基因菌株的设计更改 使其适用于毒物筛查的策略。这些变化包括可多检测的荧光素酶/绿色荧光蛋白 报告系统和通过改善化学渗透性提高了灵敏度。我们将测试我们的 通过对具有已知非优生活性的化合物进行两次小规模筛选和 生殖效应：一组来自NCI的化疗药物和一种环境毒物 环保局发来的。在项目的第二阶段(R00)，我们将把我们的系统转移到大规模、高性能- 吞吐量设置以筛选两个库，首先是ToxCast第一阶段309化学物质，然后是 大约10,000种化合物。在线虫和哺乳动物模型中首先进行二次验证将是决定性的 建立筛选的有效性，并可能导致新的减数分裂非优生化合物的鉴定。 该项目的合作基础是将我们的专业知识与环保局主要研究人员的专业知识相结合 NIEHS和巨大的培训环境使我们对这种方法的成功充满信心。 通过合作，我们将第一次能够评估我们环境的减数分裂非优生活动和 提高我们预测对哺乳动物和人类毒性的能力。