WHOLE ANIMAL DETECTION OF GENOTOXIC AGENTS

整个动物的基因毒剂检测

• 批准号: 2153981
• 负责人: PETER J. STAMBROOK
• 金额: $ 16.71万
• 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 1995-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2153981
• 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; nucleic acid probes; nucleic acid sequence; plasmids; polymerase chain reaction; stainings; toxicant screening; transcription factor; 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导致风险增加 体细胞突变，从而增加出生缺陷的风险 和癌症。 帮助更好地评估诱变行为 环境因素，该提案的目标是生产老鼠 可用作环境诱变剂的探测器。 这 检测小鼠的特征不仅可以识别诱变性 剂，但还将定义每种诱变剂的目标器官，并将 确定剂量反应的存在（或缺乏） 关系。 由于该测定使用整个动物，因此 能够区分组织特异性促诱变剂代谢 激活。 如果需要，检测到的突变的性质可以是 直接在核苷酸水平上测定。 诱变目标将 是编码 lac 阻遏物的细菌 lacI 转基因。 这 指示基因将是细菌 lacZ 转基因，其编码细菌 β-半乳糖苷酶。 转基因检测小鼠将携带一个 lacI 转基因和一个或两个 lacZ 转基因。 如果 lacI 转基因是 lacZ 转基因的功能将被抑制，并且检测器的细胞 小鼠不会对 β-半乳糖苷酶活性进行染色。 如果一个细胞发生 使 lacI 基因失活的突变，该细胞及其后代将 在未染色的背景下对 β-半乳糖苷酶进行染色。 因此，通过 单个器官和组织的切片和染色切片， 施用一种物质后，人们可以识别出哪些药剂 诱变，受诱变剂影响最大的靶器官， 给药途径取决于靶器官的分布，以及 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，扩增 通过聚合酶链式反应 (PCR) 和测序检测突变 lacI 基因 扩增产物。 最后，关系或缺乏关系可以 在发生突变的器官和组织与那些发生突变的器官和组织之间建立 随后产生肿瘤。