FLOW CYTOMETRIC ANEUGEN AND CLASTOGEN ASSAY

这个第一阶段的应用程序扩展了我们之前的研究 对自动小鼠微核(MN)分析的修改是否可以 提供有关化学物质的遗传毒性的更多机械信息 行动。体内无性激素的鉴定是人类免疫缺陷检测的重要阶段。 遗传毒性评估。非整倍体诱导剂(非整倍体)是一种 人类疾病和生殖失败的潜在源头。证据 表明异常染色体和导致 非整倍体可能在体内参与了这一过程。 肿瘤的形成和体外细胞转化。因此， 对非致癌化学品的检测和评估具有极其重要的意义。 由于微核可由无着丝粒染色体片段或由 落后于整个染色体，这个终点能够检测到 碎裂原和非整倍体一样。通过区分由整体组成的MN 染色体和片段，将有可能区分 两个人。一种自动检测非优生活性的方法将 在以下方面比现有方法有了很大的进步 周转时间、成本和可靠性。 在这笔SBIR资金的支持下，我们打算开发一种可靠的 方法区分破裂素和非整倍体诱导的MN。初步 实验表明，MN的DNA含量可用于区分 来自非优生元的碎裂源。此方法有一定的局限性，因为我们 无法区分有着丝粒的MN和没有着丝粒的MN，因此 非整倍体的明确迹象是不可能的。最近的事态发展 抗着丝粒抗体，着丝粒和端粒探针， 使非优生潜能的阳性鉴定成为可能 利用流式细胞仪技术。 在第一阶段，我们建议研究利用 FCM联合抗着丝粒抗体或着丝粒 和/或端粒探针，以识别诱导非整倍体的化学物质。 这些程序将与以前的固定和染色方法一起使用 由我们的实验室开发用于检测微核。 建议的商业应用： 检测非优生活性的自动化方法将代表着一种 在周转时间方面比现有方法有显著的进步， 成本和可靠性。拟议中的实验旨在开发 这样的自动化测试系统，并建立Litron实验室作为 专家测试设施，为行业和政府提供 高度可靠的遗传毒性检测。此外，通过将 试剂盒形式，可能实现另一种商业渠道。

This Phase I application extends our previous research by investigating whether modifications to the automated mouse micronucleus (MN) assay can provide more mechanistic information regarding a chemical's genotoxic action. The in vivo identification of aneugens is an important phase of genotoxicity evaluation. Aneuploidy inducing agents (aneugens) are a potential source of human disease and reproductive failure. Evidence suggests that abnormal chromosomes and the events giving rise to aneuploidy may be mechanistically involved in the process of in vivo tumor formation and in vitro cell transformation. Therefore, the detection and evaluation of aneugenic chemicals is of extreme importance. Since micronuclei may arise from acentric chromosome fragments or from lagging whole chromosomes, this endpoint is capable of detecting clastogens as well as aneugens. By discriminating MN comprised of whole chromosomes from fragments, it will be possible to distinguish between the two. An automated method of detecting aneugenic activity would represent a significant advance over existing methods in terms of turnaround-time, cost and reliability. With the support of this SBIR funding, we intend to develop a reliable method to discriminate clastogen versus aneugen induced MN. Preliminary experiments suggest that the DNA content of MN may be used to distinguish clastogens from aneugens. This method is somewhat limited in that we cannot distinguish MN with centromeres from those without, thus making a definitive indication of aneuploidy impossible. The recent development of anti-kinetochore antibodies, and centromeric and telomeric probes, makes the positive identification of aneugenic potential possible utilizing flow cytometry technology. During Phase I, we propose to investigate the feasibility of utilizing FCM in conjunction with anti-kinetochore antibodies, or centromeric and/or telomeric probes, to identify chemicals that induce aneuploidy. These procedures will be used with fixing and staining methods previously developed at our laboratories for the detection of micronuclei. PROPOSED COMMERCIAL APPLICATIONS: An automated method of detecting aneugenic activity would represent a significant advance over existing methods in terms of turnaround-time, cost and reliability. The experiments proposed are intended to develop such an automated test system, and establish Litron Laboratories as an expert testing facility which provides both industry and government with highly reliable genotoxicity assays. Additionally, by packaging the reagents in kit form, another commercial outlet may be realized.