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Development of an in vivo gene mutation assay using the endogenous Pig‐A gene: I. Flow cytometric detection of CD59‐negative peripheral red blood cells and CD48‐negative spleen T‐cells from the rat

The product of the phosphatidylinositol glycan complementation group A gene (Pig‐A) is involved in the synthesis of glycosylphosphatidylinositol (GPI) anchors that link various protein markers to the surface of several types of mammalian cells, including hematopoietic cells. Previous observations indicate that Pig‐A mutation results in the lack of GPI synthesis and the absence of GPI‐anchored proteins on the cell surface. As a first step in designing a rapid assay for measuring Pig‐A mutation in the rat, we developed flow cytometry (FCM) strategies for detecting GPI‐negative cells in rat peripheral blood and spleen. Anti‐CD59 was used to detect GPI‐anchored proteins on red blood cells (RBCs), and anti‐CD48 was used to detect GPI‐anchored proteins on spleen T‐cells. The spontaneous frequency of CD59‐negative RBCs in five male F344 rats ranged from 1 × 10−6 to 27 × 10−6. In contrast, treatment of five rats with three doses of 40 mg/kg N‐ethyl‐N‐nitrosourea (ENU) increased the frequency of CD59‐negative RBCs to 183 × 10−6 to 249 × 10−6 at 2 weeks and to 329 × 10−6 to 413 × 10−6 at 4 weeks after dosing. In the same 4‐week posttreatment rats, the frequency of CD48‐negative T‐cells was 11 × 10−6 to 16 × 10−6 in control rats and 194 × 10−6 to 473 × 10−6 in ENU‐treated rats. The frequencies of GPI‐deficient cells were similar for RBCs and spleen T‐cells. These results indicate that FCM detection of GPI‐linked markers may form the basis for a rapid in vivo mutation assay. Although RBCs may be useful for a minimally invasive assay, T‐cells are a promising tissue for both detecting GPI‐deficient cells and confirming that Pig‐A gene mutation is the cause of the phenotype. Environ. Mol. Mutagen., Published 2008 Wiley‐Liss, Inc.