IN VITRO AND IN VIVO MUTAGENESIS TESTING--FLOW CYTOMETRY

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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.