Automation of the in vitro Micronucleus Assay

体外微核测定的自动化

• 批准号: 6442812
• 负责人: STEPHEN D DERTINGER
• 金额: $ 9.86万
• 依托单位: LITRON LABORATORIES, LTD.
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-06-01 至 2002-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6442812
• 关键词: DNA damage; apoptosis; bioassay; biomedical automation; biotechnology; cell cycle; cell line; chemical hypersensitivity; flow cytometry; fluorescence microscopy; method development; micronucleus; mutagen testing; terminal nick end labeling

The in vitro nucleus test is increasingly being used as an alternative to the more costly and time consuming in vitro chromosome aberration assay as a screen for detecting a chemical's genotoxic potential (aneugenic or clastogenic activity which are associated with disorders such as Down's Syndrome, cancers, etc.) Micronucleus measurements are made using the time-consuming and tedious process of microscopic inspection. No definitive and widely-accepted automated approach has come into use. During Phase I, we will investigate the use of flow cytometry to enumerate micronuclei in mouse lymphoma L5178 cells. This cell line is used in a commonly performed forward mutation assays (i.e., thymidine kinase locus) required by regulatory agencies world-wide. These cells offer the potential to develop an assay where the cells are treated and subsequently split between micronucleus and mutation endpoints, thereby minimizing plasticware, test article, and time requirements. Studies will focus on identifying gating and fluorescent staining procedures to discriminate bodies and mitotic chromosomes from true micronucleus events. Long range, the automated scoring procedures developed can be extended to the analysis of micronuclei in human lymphocytes. Some of these applications may include: 1) assessing the genotoxicity of pharmaceuticals. 2) identifying the individuals hypersensitive in particular genotoxin, 3) evaluating compounds or diets protective against genetic damage and 4) measurement of DNA damage in a population following a radiation and/or chemical accident. PROPOSED COMMERCIAL APPLICATION: The successful development of this methodology will enable Litron Laboratories to become an expert testing facility, capable of high throughput screening which identifies agents that cause cytogenetic damage. Furthermore, the extension of this methodology to the analysis of human blood lymphocytes and into kit format represents other significant areas of commercial opportunity.

体外细胞核试验越来越多地被用作更昂贵和耗时的体外染色体畸变试验的替代方法，作为检测化学品遗传毒性潜力（与唐氏综合征、癌症等疾病相关的非整倍体或致染色体断裂活性）的筛选。微核测量是使用耗时和繁琐的显微镜检查过程。没有明确的和广泛接受的自动化方法已经投入使用。在I期，我们将研究使用流式细胞术计数小鼠淋巴瘤L5178细胞中的微核。该细胞系用于通常进行的正向突变测定（即，胸苷激酶基因座）。这些细胞提供了开发一种试验的可能性，其中细胞经过处理，随后在微核和突变终点之间进行分离，从而最大限度地减少塑料器皿、供试品和时间要求。研究将侧重于确定门控和荧光染色程序，以区分体和有丝分裂染色体与真正的微核事件。开发的自动评分程序可扩展到人淋巴细胞微核分析。这些应用中的一些可能包括：1）评估药物的遗传毒性。2)鉴定特别是遗传毒素过敏的个体，3）评估防止遗传损伤的化合物或饮食，和4）测量辐射和/或化学事故后人群中的DNA损伤。拟定商业应用：这种方法的成功开发将使Litron实验室成为一个专家测试设施，能够高通量筛选，确定代理，造成细胞遗传学损伤。此外，将该方法扩展到人血淋巴细胞分析和试剂盒形式代表了其他重要的商业机会领域。