MULTILOCUS MICROTITER IN SITU MOUSE LYMPHOMA ASSAY

建议研究开发一种新的哺乳动物细胞诱变试验 其中p53抑癌基因的突变或丢失可能是 使用L5178y/tk+/-3.7.2C电池进行高效且经济的评估， 最近发现它们是p53和tk的杂合子。一个 突变菌落大小双峰分布的另一种假设 在小鼠淋巴瘤试验(MLA)中观察到的大Lambda 菌落突变是由一个等位基因tk+的突变或丢失引起的，并且 小西格玛菌落突变是由两个等位基因突变或丢失引起的， TK+和P53-，均位于3.7.2C染色体11b远端15%处 因此，在tk基因座突变的细胞的生长速度取决于 显性阴性的P53基因是保留还是丢失。恢复 Sigma菌落突变体的数量在原位MLA(ISMLA)中大大增强 哪些细胞在化学暴露后被克隆，以及初步结果 这表明，如果单元格是 克隆在微滴度(MT)平板上而不是在软琼脂中。在第一阶段， 将定义经济多位点(ML)MT-ISMLA，其中P53 Lambda和Sigma菌落突变体的表型将通过 免疫组织化学染色；它将在阶段中自动进行验证 一种准确和经济的MLMT ISMLA，它可以评估突变和 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关 检测诱变和碎裂发生机制的试验， 预测癌症发生，评估用于监管的化学品 提交材料和风险评估。 建议的商业应用： ML-MT ISMLA在它被精确地 由监管机构定义、自动化、验证和接受。 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检测，举办培训研讨会， 提供MLA菌落P53免疫组织化学染色服务、咨询 与使用化验和解释其结果有关的服务， 以及用于该检测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.