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

非整倍体，或细胞中染色体数目异常，起源于染色体分离错误。 在细胞分裂、有丝分裂和减数分裂这两个高度调节的程序中，染色体的结构发生变化。的 减数分裂起源的非整倍性的后果是毁灭性的，因为它们导致35%的临床 据统计，4%的死产、20%的出生缺陷和几乎所有的生殖细胞肿瘤都与流产有关。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， 塑料、杀虫剂和抗焦虑药物等多种化合物已被证明会引起减数分裂。 哺乳动物的非整倍性 尽管减数分裂非整倍体对健康有严重影响，但环境毒物的筛选 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部分地显著不同于有丝分裂，不能有效地 在体外重演，并在哺乳动物发育早期在胚胎发育的范围内启动。 生殖腺因此，我们目前缺乏有效和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性的影响。 在这个应用中，我们提出了一种高通量筛选方法， 秀丽隐杆线虫的显著生物学特征，作为减数分裂模式 系统显示出与人类高度的保守性。为此，我们正制订一项策略， 开发用于分离减数分裂期间染色体分离缺陷的突变体。在K99部分 在项目的最后阶段，我们将完成C.本发明所用的线虫转基因株 使其适合于毒物筛选的策略。这些变化包括多个可测定的荧光素酶/GFP 报告系统，并通过改善化学渗透提高灵敏度。我们将测试我们的有效性 方法通过对具有已知非整倍体活性的化合物进行两次小规模筛选， 生殖影响：一组来自NCI的化疗药物和一种环境毒物 从EPA。在项目的第二阶段（R 00），我们将把我们的系统推向大规模，高- 使用通量设置筛选两个文库，首先是Toxcast I期309化学品，然后是Tox 21文库。 大约一万种化合物二次验证首先在C中进行。在哺乳动物模型中， 建立筛选的有效性，并可能导致鉴定新的减数分裂非整倍体化合物。 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。 总之，我们将能够第一次评估我们环境的减数分裂非整倍性活动， 提高我们预测对哺乳动物和人类毒性的能力。

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.