WHOLE ANIMAL DETECTION OF GENOTOXIC AGENTS

整个动物的基因毒剂检测

• 批准号: 3253469
• 负责人: PETER J. STAMBROOK
• 金额: $ 14.44万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-08-01 至 1994-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3253469
• 关键词: bacterial genetics; beta galactosidase; bioassay; diagnosis design /evaluation; environmental contamination; genetic promoter element; genetically modified animals; histochemistry /cytochemistry; laboratory mouse; mutagen testing; natural gene amplification; nucleic acid probes; plasmids; polymerase chain reaction; stainings; toxicant screening; transposon /insertion element

Exposure to environmental mutagens contributes to an increased risk of somatic mutation, and consequently, an increased risk of birth defects and of cancer. To help better assess the mutagenic behavior of environmental agents, the objective of this proposal is to produce mice that can be used as detectors of environmental mutagens. The characteristics of the detector mice will not only identify mutagenic agents but will also define the target organs for each mutagen, and will establish the existence (or lack thereof) of a dose-response relationship. Since this assay utilizes whole animals, it will be capable of distinguishing tissue-specific promutagen metabolic activation. If desired, the nature of the detected mutations can be directly determined at the nucleotide level. The mutagenesis target will be a bacterial lacI transgene which encodes the lac repressor. The indicator gene will be a bacterial lacZ transgene which encodes bacterial beta-galactosidase. The transgenic detector mice will carry a single lacI transgene and one or two lacZ transgenes. If the lacI transgene is functional the lacZ transgenes will be repressed, and cells of detector mice will not stain for beta-galactosidase activity. If a cell incurs a mutation that inactivates the lacI gene, that cell and its progeny will stain for beta-galactosidase against an unstained background. Thus, by sectioning and staining sections of individual organs and tissues, following administration of a substance, one can identify agents that are mutagenic, the target organs most affected by that mutagen, the effect of route of administration upon the distribution of target organs, and the relative potency of the agent. The nature of the mutation can be determined by extracting DNA from the affected cells, amplifying the mutant lacI gene by the polymerase chain reaction (PCR), and sequencing the amplified product. Lastly, a relationship, or absence thereof, can be established between organs and tissues that incur mutations and those that later produce tumors.

暴露于环境诱变剂会增加 体细胞突变，从而增加出生缺陷的风险 和癌症。 为了帮助更好地评估 环境因子，本提案的目的是生产小鼠 可以用作环境诱变剂的检测器。 的 检测小鼠的特征不仅可以识别诱变性， 但也将确定每种诱变剂的靶器官，并将 确定存在（或缺乏）剂量反应 关系 由于该试验使用整个动物，因此将 能够区分组织特异性前诱变剂代谢 activation. 如果需要，可以检测检测到的突变的性质。 直接在核苷酸水平测定。 诱变目标将 是编码lac阻遏物的细菌lacI转基因。 的 指示基因将是编码细菌lacZ转基因， β-半乳糖苷酶。 转基因探测鼠将携带一个 lacI转基因和一个或两个lacZ转基因。 如果lacI转基因 功能性的lacZ转基因将被抑制，并且检测器的细胞 小鼠将不会对β-半乳糖苷酶活性染色。 如果一个细胞引发 当突变使lacI基因失活时，该细胞及其后代将 针对未染色背景进行β-半乳糖苷酶染色。 因此，由 对单个器官和组织切片并染色， 在给予物质后，可以鉴定出 诱变，受诱变剂影响最大的靶器官， 给药途径对靶器官分布的影响， 药剂的相对效力。 突变的性质可以是 通过从受影响的细胞中提取DNA、放大DNA来确定 通过聚合酶链反应（PCR）获得突变的lacI基因，并测序 放大的产品。 最后，一种关系，或没有关系，可以 在引起突变的器官和组织之间建立， 然后产生肿瘤