Flow Cytometric Analysis of Genotoxicity

遗传毒性的流式细胞术分析

• 批准号: 6484490
• 负责人: JOHNATHAN C SHARPE
• 金额: $ 42.85万
• 依托单位: CYTOMATION GTX, INC.
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-18 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6484490
• 关键词: DNA damage; biotechnology; cell line; cytotoxicity; drug adverse effect; flow cytometry; gene mutation; method development; surface antigens

The overall objective of this proposal is to develop a flow cytometry- based assay utilizing optimized flow cytometry instrumentation and cultured mammalian cells that can be routinely employed by pharmaceutical and other companies to measure the mutagenic potential of chemicals and drugs. The assay is based on a Chinese hamster ovary- human hybrid cell line, CHO AL, which contains human chromosome 11. The principal target for mutation is the gene for the CD59 antigen which is encoded on chromosome 11. The hypotheses are(1) that mutations in chromosome 11 can be detected accurately, sensitively and rapidly by flow cytometry measurement of the loss or presence of surface antigens encoded by chromosome 11 and (2) that this mutation assay system can be developed so that it could be used routinely to determine the genotoxicity of physical and chemical agents. Specific Aim 1 is to build a relatively inexpensive benchtop flow cytometer optimized for analysis of antigen loss in mammalian cells. Specific Aim 2 is to validate the flow cytometry assay for detecting mutations in CHO AL cells by measuring the mutation dose response from a panel of 25 physical and chemical agents at a range of doses. Independent studies will be done at an off-site laboratory. Specific Aim 3 is to extend the assay to include the analysis of intragenic and chromosomal mutations by measuring the presence or absence of multiple antibodies bound to different cell surface antigens encoded by separate genes on human chromosome 11. The development of this assay system would allow for rapid, accurate, relatively cheap but informative screening of pharmaceuticals for genotoxicity and substantially reduce the cost of carrying out mutation analysis.

本提案的总体目标是开发一种基于流式细胞术的试验，该试验利用优化的流式细胞术仪器和培养的哺乳动物细胞，可由制药公司和其他公司常规使用，以测量化学品和药物的致突变潜力。该试验基于中国仓鼠卵巢-人杂交细胞系CHO AL，其含有人11号染色体。突变的主要靶点是在11号染色体上编码的CD 59抗原的基因。假设是：（1）通过流式细胞术测量11号染色体编码的表面抗原的缺失或存在，可以准确、灵敏和快速地检测11号染色体中的突变;（2）可以开发这种突变检测系统，以便其可以常规用于测定物理和化学试剂的遗传毒性。具体目标1是建立一个相对便宜的台式流式细胞仪优化分析的抗原损失在哺乳动物细胞。具体目标2是通过测量一组25种物理和化学试剂在一系列剂量下的突变剂量反应，验证用于检测CHO AL细胞突变的流式细胞术试验。独立研究将在场外实验室进行。具体目标3是通过测量是否存在与人11号染色体上不同基因编码的不同细胞表面抗原结合的多种抗体，将试验扩展至包括基因内突变和染色体突变分析。 该分析系统的开发将允许快速、准确、相对便宜但信息丰富的药物遗传毒性筛选，并大大降低进行突变分析的成本。