Abstract
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 × 10^−6^ to 4.8 × 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. Environ. Mol. Mutagen., 2008. Published 2008 Wiley‐Liss, Inc.