IN VITRO AND IN VIVO MUTAGENESIS TESTING--FLOW CYTOMETRY

发展了一种检测单细胞DNA损伤的新方法。 流式细胞仪检测细胞数。该方法是基于免疫荧光的 用特异性抗体检测DNA复制 5-溴脱氧尿苷(BrdUrd)。我们正在将这种方法扩展到 通过选择耐药性来检测突变(例如， 硫代鸟嘌呤抗性)细胞，该细胞将在 毒品的存在。细胞的特异性抗体染色 对于BrdUrd加上荧光标记，可以在流中对其进行分析 细胞仪，随后在培养中进行生长和选择。 我们正在利用单抗和免疫荧光放大技术 提高灵敏度和信号的技术：必要的噪声 检测极少量的阳性细胞 诱变试验。 还提出了采用该方法学进行基因分析的方法。 由染色体异常或相关因素引起的杂合子分离 可能表明化合物的致畸行为。 我们预计这些技术将适用于评估 体内外的DNA损伤和诱变。在后一种情况下，这 将提供快速、短期的诱变剂和致畸剂测试 系统现在不能接受这种分析。

A new method has been developed for the detection of DNA damage in single cells by flow cytometry. The method is based on the immunofluorescent detection of DNA replication with specific antibodies against 5-Bromodeoxyuridine (BrdUrd). We are extending this methodology to the detection of mutations by the selection of drug resistant (e. g. thioguanine resistance) cells which will incorporate bromedeoxyuridine in the presence of the drug. Staining of the cells with antibodies specific for BrdUrd plus a fluorescent tag enables their analysis in a flow cytometer, subsequent to growth and selection in culture. We are utilizing monoclonal antibodies and immunofluorescent amplification techniques to increase the sensitivity and signal: noise necessary to detect the extremely small numbers of positive cells required for mutagenesis assays. Methods are also proposed to employ the methodology for analysis of gene segregation from heterozygotes caused by chromosome aberrations or related phemonena which may be indicative of teratogenic behavior by compounds. We anticipate that these techniques will be applicable to the assessment of DNA damage and mutagenesis in vitro and vivo. In the latter case, this would provide for rapid, short-term testing of mutagens and teratogens in systems not now amenable to this kind of analysis.