Use of somatic cell hybrids for quantitation of mutagenesis: reduction in background mutants by fluorescence-activated cell sorting (FACS).
Environmentally induced mutations, especially those involving large scale genetic damage such as deletions and chromosome loss, are of central importance in the production of human genetic disease and cancer. We have developed a methodology, the AL assay, that permits detection of such extensive genetic changes which often escape detection in other systems in which they are lethal. The AL assay employs a human-Chinese hamster ovary cell hybrid that retains a single human chromosome, number 11. A set of specific cell surface antigens result from genes located on opposite arms of this chromosome. Exposure to mutagens produces mutants which form colonies in the presence of complement and specific antiserum that kill nonmutant cells. The frequency and pattern of marker loss provides a measure of single gene mutation, large and small deletion, and loss of the entire chromosome 11. We have employed the indirect fluorescein conjugated isothiocyanate (FITC) technique and fluorescence-activated cell sorting (FACS) to remove spontaneous mutants from the initial population. The 100-fold reduction in background thus far achieved should allow accurate analysis of mutation by ionizing radiation at doses of less than 10 rad.
D000818:Animals; D002460:Cell Line; D002469:Cell Separation / Q000379:methods; D003165:Complement System Proteins / Q000276:immunology; D006224:Cricetinae; D003412:Cricetulus; D003602:Cytotoxicity, Immunologic; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D006822:Hybrid Cells / Q000166:cytology*; D009154:Mutation; D010053:Ovary; D013861:Thiocyanates
D005452:Fluoresceins; D005456:Fluorescent Dyes; D013861:Thiocyanates; D003165:Complement System Proteins; D016650:Fluorescein-5-isothiocyanate