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An assay for loss of heterozygosity in vivo at the Dlb-1 locus.

Loss of heterozygosity (LOH) is a frequent event in many tumours, resulting in the loss of tumour suppressor genes and ultimately magnifying the number of cells with a mutant phenotype. We have used the Dlb-1 locus as a simple quantitative assay for LOH in vivo. Mutations of the dominant Dlb-1b allele are readily detected in heterozygous (Dlb-1a/Dlb-1b) mice by the loss of histochemical staining, which results in unstained, white (Dlb-1a/Dlb-1-) ribbons on a stained background. Such ribbons are extremely rare in untreated C57BL mice which are homozygous for the dominant allele, as would be expected when two independent mutations are required. To test for LOH, we first treated the animals with a high dose of ethylnitrosourea (ENU) which induces many mutations and thus many heterozygous cells, and allowed 2 weeks for gene expression. Then the animals were treated with the test agent to determine if it could cause LOH and thus convert heterozygous mutant cells, which would not produce detectable ribbons, into homozygotes that would produce white, non-staining ribbons. Treatment with ENU alone produced a low, but detectable, frequency of mutant ribbons. Treatment with X-rays alone produced no detectable increase in the frequency of mutant ribbons. Combinations of these treatments produced additive effects, thus showing that no significant LOH was induced. The additivity of the two equal ENU treatments was unexpected, since double mutants should increase as the square of the mutant frequency.(ABSTRACT TRUNCATED AT 250 WORDS)