BACKGROUND:Loss of heterozygosity (LOH) contributes to many cancers, but the rate at which these events occur in normal cells of the body is not clear. LOH would be detectable in diverse cell types in the body if this event were to confer an obvious cellular phenotype. Mice that carry two different fluorescent protein genes as alleles of a locus would seem to be a useful tool for addressing this issue because LOH would change a cell's phenotype from dichromatic to monochromatic. In addition, LOH caused by mitotic crossing over might be discernable in tissues because this event produces a pair of neighboring monochromatic cells that are different colors.RESULTS:As a step in assessing the utility of this approach, we derived primary embryonic fibroblast populations and embryonic stem cell lines from mice that carried two different fluorescent protein genes as alleles at the chromosome 6 locus, ROSA26. Fluorescence activated cell sorting (FACS) showed that the vast majority of cells in each line expressed the two marker proteins at similar levels, and that populations exhibited expression noise similar to that seen in bacteria and yeast. Cells with a monochromatic phenotype were present at frequencies on the order of 10(-4) and appeared to be produced at a rate of approximately 10(-5) variant cells per mitosis. 45 of 45 stably monochromatic ES cell clones exhibited loss of the expected allele at the ROSA26 locus. More than half of these clones retained heterozygosity at a locus between ROSA26 and the centromere. Other clones exhibited LOH near the centromere, but were disomic for chromosome 6.CONCLUSION:Allelic fluorescent markers allowed LOH at the ROSA26 locus to be detected by FACS. LOH at this locus was usually not accompanied by LOH near the centromere, suggesting that mitotic recombination was the major cause of ROSA26 LOH. Dichromatic mouse embryonic cells provide a novel system for studying genetic/karyotypic stability and factors influencing expression from allelic genes. Similar approaches will allow these phenomena to be studied in tissues.

Exposure to inorganic arsenic in drinking water is linked to cancer in humans, but the mechanism of arsenic-induced cancer is not clear. Arsenic is not a powerful point mutagen, but can cause chromosome malsegregation and mitotic recombination, two events that can cause loss of tumor suppressor alleles and thereby contribute to the evolution of cancerous cells. To determine whether arsenic increases the frequency of allele loss due to either malsegregation or mitotic recombination in vivo, Apr(+/-) hybrid mice were exposed to sodium arsenite (10 mg/L) in their drinking water for 10 weeks. To determine whether arsenic enhances the action of a known mutagen, half of the arsenic-treated mice were exposed to benzo[a]pyrene (BaP) for 8 weeks by skin painting (500 nmoles/ week). Cells were taken from painted dorsal skin and cultured in the presence of 2,6-diaminopurine (DAP), to select colonies lacking adenosine phosphoribosyl transferase (Aprt) activity. The frequency of DAP-resistant (DAp(r)) colonies varied substantially within the treatment groups, but there was no significant difference between the groups. Analysis of DNA from DApr colonies suggested that mitotic recombination contributed to the loss of wild-type Aprt allele. Whether arsenic or BaP enhanced or diminished the frequency of this process could not be deduced from these data.

Exposure to inorganic arsenic in drinking water is linked to cancer in humans, but the mechanism of arsenic-induced cancer is not clear. Arsenic is not a powerful point mutagen, but can cause chromosome malsegregation and mitotic recombination, two events that can cause loss of tumor suppressor alleles and thereby contribute to the evolution of cancerous cells. To determine whether arsenic increases the frequency of allele loss due to either malsegregation or mitotic recombination in vivo, Aprt +/− hybrid mice were exposed to sodium arsenite (10 mg/L) in their drinking water for 10 weeks. To determine whether arsenic enhances the action of a known mutagen, half of the arsenic-treated mice were exposed to benzo[ a ]pyrene (BaP) for 8 weeks by skin painting (500 nmoles/week). Cells were taken from painted dorsal skin and cultured in the presence of 2,6-diaminopurine (DAP), to select colonies lacking adenosine phosphoribosyl transferase (Aprt) activity. The frequency of DAP-resistant (DAP r ) colonies varied substantially within the treatment groups, but there was no significant difference between the groups. Analysis of DNA from DAP r colonies suggested that mitotic recombination contributed to the loss of wild-type Aprt allele. Whether arsenic or BaP enhanced or diminished the frequency of this process could not be deduced from these data. Environ. Mol. Mutagen., 2006. © 2006 Wiley-Liss, Inc.

Harlequin (Hq) mice develop ataxia due to an X-linked recessive mutation in the gene encoding apoptosis-inducing factor (Aif). Brain cells in Hq mice contain the modified base 8-hydroxydeoxyguanosine (8-OHdG), suggesting that the defect in Aif causes increased DNA oxidation in these cells. Because oxidative damage is mutagenic, Hq mice might suffer increased mutation in the brain. To examine this possibility, mutation in the brain was assessed using the Tg(βA-G11PLAP) mouse model, which allows mutant cells to be visualized in tissue sections in situ. Hq mice exhibited more and larger patches of PLAP positive tissue in the brain. PLAP+ cells were observed in all areas of the brain. No increase in the number of PLAP+ cells was seen in three other tissues, suggesting that the effect of Aif deficiency on mutation was specific to brain.