Development of an in vivo gene mutation assay using the endogenous Pig-A gene: II. Selection of Pig-A mutant rat spleen T-cells with proaerolysin and sequencing Pig-A cDNA from the mutants.

We previously reported that rat spleen T-cells and peripheral red blood cells that are deficient in glycosylphosphatidylinositol (GPI) synthesis [presumed mutants for the phosphatidylinositol glycan complementation group A gene (Pig-A)] could be detected by flow cytometry (FCM) as cells negative for GPI-linked markers (CD48 and CD59, respectively). To establish this procedure as a rapid in vivo gene mutation assay, we have examined the Pig-A gene of GPI-deficient rat spleen T-cells for DNA sequence alterations. Splenocytes were isolated from male F344 rats, primed with ionomycin and phorbol-12-myristate-13-acetate, and seeded at limiting-dilution into 96-well plates. To select for GPI-deficient T-cells, the cells were cultured for 10 days in a medium containing rat T-STIM and 2 nM proaerolysin (ProAER). The frequency of ProAER-resistant (ProAER(r)) spleen T-cells from control rats ranged from 1.3 x 10(-6) to 4.8 x 10(-6), while administration of three doses of 40 mg/kg N-ethyl-N-nitrosourea increased the frequency of ProAER(r) T-cells 100-fold at 4 weeks after dosing. FCM analysis of the cells in ProAER(r) clones revealed that they were CD48-negative, and thus presumably GPI-deficient. Sequencing of Pig-A cDNA from six ProAER(r) clones indicated that they all contained alterations in the Pig-A protein coding sequence; five had base pair substitutions and one had multiple exons deleted. These results indicate that GPI-deficient spleen T-cells are Pig-A gene mutants and support the use of FCM analysis of GPI-deficient cells as a rapid assay for measuring in vivo gene mutation. Published 2008 Wiley-Liss, Inc.