Automation of the in vitro Micronucleus Assay

体外核试验越来越多地被用作昂贵和耗时的体外染色体畸变测定的替代品，作为检测化学物质遗传毒性潜力（与唐氏综合症、癌症等疾病相关的非优生或致裂活性）的筛选。微核测量使用耗时和繁琐的显微镜检查过程。目前还没有明确的、被广泛接受的自动化方法投入使用。在I期，我们将研究使用流式细胞术枚举小鼠淋巴瘤L5178细胞中的微核。该细胞系用于世界各地监管机构要求的常用正向突变测定（即胸苷激酶位点）。这些细胞提供了开发一种检测方法的潜力，在这种方法中，细胞经过处理并随后在微核和突变端点之间分裂，从而最大限度地减少塑料器皿、测试品和时间要求。研究将集中在鉴别门控和荧光染色程序，以区分实体和有丝分裂染色体从真正的微核事件。从长远来看，开发的自动评分程序可以扩展到分析人类淋巴细胞中的微核。其中一些应用可能包括：1)评估药物的遗传毒性。2)识别对特定基因毒素过敏的个体，3)评估防止基因损伤的化合物或饮食，4)测量辐射和/或化学事故后人群的DNA损伤。建议的商业应用：该方法的成功开发将使Litron实验室成为专家测试机构，能够进行高通量筛选，识别导致细胞遗传损伤的药物。此外，将这种方法扩展到人类血液淋巴细胞的分析和试剂盒格式代表了其他重要的商业机会领域。

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.