MULTILOCUS MICROTITER IN SITU MOUSE LYMPHOMA ASSAY

本研究旨在开发一种新的哺乳动物细胞诱变检测方法 其中p53肿瘤抑制基因的突变或缺失可 使用L5178 y/tk+/-3.7.2C细胞有效和经济地评估， 最近发现其对于p53以及对于TK是杂合的。一个 突变菌落大小双峰分布的另一种假设 小鼠淋巴瘤试验（MLA）中观察到的结果表明，大λ 菌落突变体由一个等位基因tk+突变或缺失引起， 小σ菌落突变体由两个等位基因突变或缺失产生， tk+和p53-，均见于3.7.2C中11b染色体远端15%处 细胞;因此，在tk基因座突变的细胞的生长速率取决于 显性负性p53基因型是保留还是丢失。恢复 在原位MLA（ISMLA）中， 哪些细胞在化学暴露后被克隆，以及初步结果 这表明，如果细胞被 在微量滴定（MT）板中而不是在软琼脂中克隆。在第一阶段， 将定义经济的多位点（ML）MT-ISMLA，其中p53 λ和σ菌落突变体的表型将通过 免疫组织化学染色;将在第三阶段自动化并验证 二.一个准确和经济的MLMT ISMLA，评估突变和 p53和tk的杂合性丢失将显著更多 与传统的MLA协议和许多其他短期协议相比， 检测诱变和染色体断裂机制的试验， 预测致癌作用，评估化学品， 提交和风险评估。 拟定商业应用： ML-MT ISMLA应该在精确测量后有很高的需求。 定义、自动化、验证并被监管机构接受。 潜在的商业应用包括营销MT-ML ISMLA研究 并向政府和行业进行测试，举办培训讲习班， 提供MLA集落p53免疫组化染色服务，咨询 与检测试剂盒使用及其结果解释相关的服务， 以及用于该分析的图像分析系统的商业化。

Research is proposed to develop a novel mammalian cell mutagenesis assay in which mutation or loss of the p53 tumor suppressor gene can be efficiently and economically assessed using L5178y/tk+/-3.7.2C cells, which were recently found to be heterozygous for p53 as well as for tk. An alternate hypothesis for the bimodal distribution of mutant colony sizes observed in the mouse lymphoma assay (MLA) suggests that large lambda colony mutants result from mutation or loss of one allele, tk+, and that small sigma colony mutants result from mutation or loss of two alleles, tk+ and p53-, both found on the distal 15% of chromosome 11b In 3.7.2C cells; thus, the growth rates of cells mutated at the tk locus depend upon whether the dominant negative p53 genotype is retained or lost. Recovery of sigma colony mutants is greatly enhanced In an in situ MLA (ISMLA) in which cells are cloned after chemical exposure, and preliminary results suggest that even greater precision may be obtained if the cells are cloned in microtiter (MT) plates instead of in soft agar. In Phase I, an economical multi-locus (ML) MT-ISMLA will be defined in which the p53 phenotype of lambda and sigma colony mutants will be assessed by immunohistochemical staining; it will be automated and validated in Phase II. An accurate and economical MLMT ISMLA which assesses mutations and loss of heterozygosity at p53 as well as tk will be significantly more relevant than conventional MLA protocols and numerous other short-term tests for examining mechanisms of mutagenesis and clastogenesis, predicting carcinogenesis, evaluating chemicals for regulatory submissions, and risk assessment. PROPOSED COMMERCIAL APPLICATION: The ML-MT ISMLA should be in high demand after it has been precisely defined, automated, validated, and accepted by regulatory agencies. Potential commercial applications include marketing MT-ML ISMLA research and testing to the government and industry, holding training workshops, offering MLA colony p53 immunohistochemical staining services, consulting services related to use of the assay and interpretation of its results, and commercialization of an image analysis system for this assay.