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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

基本信息

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
项目类别：
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

项目摘要

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，或细胞中染色体数量异常，源于染色体分离错误细胞分裂、有丝分裂和减数分裂这两个高度调控的程序中的染色体。这减数分裂起源的非整倍体的后果是毁灭性的，因为它们导致了 35% 的临床已知的流产、4% 的死产、20% 的出生缺陷以及几乎所有生殖细胞肿瘤。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，如塑料、杀虫剂和抗焦虑药物等多种化合物已被证明会导致减数分裂哺乳动物的非整倍体。尽管减数分裂非整倍体会带来严重的健康后果，但环境毒物的筛查因为它们破坏减数分裂和导致非整倍体的能力已经严重滞后。这个问题是固有的减数分裂程序的复杂性与有丝分裂部分显着不同，无法有效地在体外重现并在哺乳动物胚胎发育过程中的早期开始性腺。因此，我们目前缺乏有效、全面地询问我们的化学物质的工具。环境对减数分裂衍生的非整倍体的影响。在此应用中，我们建议开发一种高通量筛选方法，该方法利用线虫秀丽隐杆线虫的显着生物学特征，作为减数分裂模型系统显示出对人类的高度保护。为了实现这一目标，我们正在制定一项战略开发用于分离减数分裂过程中染色体分离缺陷的突变体。在K99部分项目完成后，我们将完成该项目中使用的秀丽隐杆线虫转基因菌株的设计变更使其适合毒物筛查的策略。这些变化包括可多重检测的荧光素酶/GFP 报告系统并通过改善化学渗透来提高灵敏度。我们将测试我们的有效性方法是对具有已知非整体活性的化合物进行两次小规模筛选，生殖影响：NCI 的一套化疗药物和一种环境毒物来自美国环保局。在该项目的第二阶段（R00），我们将把我们的系统转移到大规模、高吞吐量设置可筛选两个文库，首先是 Toxcast Phase I 309 化学品，然后是 Tox21 文库约 10,000 种化合物。首先在秀丽隐杆线虫和哺乳动物模型中进行二次验证将得出结论确定筛选的有效性，并可能导致新型减数分裂非整倍体化合物的鉴定。该项目的合作基础将我们的专业知识与 EPA 的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。在一起，我们将首次能够评估我们环境的减数分裂非整体活动，并提高我们预测对哺乳动物和人类的毒性的能力。