Use of somatic cell hybrids for quantitation of mutagenesis: Reduction in background mutants by fluorescence‐activated cell sorting (FACS)

Abstract

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.

Measurement of low levels of x-ray mutagenesis in relation to human disease.

“…Further steps planned in these developments should include elimination of the bulk of background mutations, as described in preliminary publications (33), increasing the sensitivity by increase in the number of markers utilized so that the target size is increased, addition of translocations to the scored mutants, analysis of repeated doses and dose rate effects like those reported by Little and Thilly (13,28), and increase in rapidity, convenience, and economy of the procedure. It appears feasible to reach a sensitivity capable of direct detection of mutagenic effects in the neighborhood of background radiation and spontaneous cellular metabolic processes.…”

Induction of somatic mutations by low-dose X-rays: the challenge in recognizing radiation-induced events

“…Mutation spectrum analysis revealed that the major mutations seen among the spontaneous mutations in this cell line were large deletions, where both the DXS1048 and DXS1194 markers were lost. This may be due to the instability of the human X-chromosome in rodent cells [ 15 – 17 ]. The mutant frequency with double-negative type mutations did not change, even when cells were irradiated (Fig.…”

“…In this cell line, cell viability is independent of any mutagenic events occurring on the human X-chromosome; thus, this cell line is expected to tolerate a higher mutant frequency than conventional systems. In addition, because human chromosomes transferred into rodent cells are known to be unstable [ 15 – 17 ], this aspect of the cells could enhance its mutator phenotype. Indeed, this cell system was found to exhibit a more than 50-fold increase in the radiation-induced mutant frequency when compared with conventional assay systems [ 14 , 18 ].…”

Telomeric repeat organization—a comparative in situ study between man and rodent

“…The two mouse cell lines used were O22 (Lee and Singer, 1982), containing a single African green monkey (AGM) chromosome on a mouse background, and CY10, containing a human chromosome 16 on a mouse background (kindly provided by Bryone Kuss). The two hamster cell lines used were ALJ2 (Waldren et al, 1984), containing human chromosome 11 on a hamster background, and GM7297, containing human chromosomes 3 and X on a hamster background. All cells were grown and harvested according to standard methodology.…”