Flow Cytometric Analysis of Genotoxicity

本提案的总体目标是利用优化的流式细胞仪仪器和培养的哺乳动物细胞开发一种基于流式细胞仪的检测方法，这种方法可以被制药公司和其他公司常规地用于测量化学物质和药物的致突变潜力。该试验是基于含有人类11号染色体的中国仓鼠卵巢-人类杂交细胞系CHO AL。突变的主要目标是编码在11号染色体上的CD59抗原基因。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、灵敏和快速地检测到11号染色体的突变；(2)可以开发出这种突变测定系统，以便可以常规地用于确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，优化用于分析哺乳动物细胞中的抗原损失。具体目标2是通过测量25种物理和化学试剂在一定剂量范围内的突变剂量反应，验证流式细胞术检测CHO AL细胞突变的效果。独立研究将在校外实验室进行。具体目标3是通过测量与人类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的存在或不存在，将该检测扩展到包括基因内和染色体突变的分析。该检测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。

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.