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IN VITRO AND IN VIVO MUTAGENESIS TESTING--FLOW CYTOMETRY

体外和体内诱变测试--流式细胞术

基本信息

批准号：
3250668
负责人：
HOWARD G GRATZNER
金额：
$ 7.69万
依托单位：
UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
1983
资助国家：
美国
起止时间：
1983-09-28 至 1986-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3250668
关键词：
6 thioguanine DNA bromodeoxyuridine chromosome disorders drug resistance flow cytometry heterozygote immunofluorescence technique monoclonal antibody mutagen testing mutagens occupational hazard teratogens

项目摘要

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.

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。