Abstract LB-153: Comparison of the Xk and Pig-a genes as markers for mutations in red cells from mice.

Abstract

Abstract For very few ‘sentinel’ genes, it is possible to screen for phenotypic variants. We previously showed that the X-linked PIG-A and XK genes can be used to measure spontaneous mutations in human erythrocytes using flow cytometry, and others have performed similar work using Pig-a in mice. Here we have shown that Xk can be used in mice as well. Pig-a is essential for the biosynthesis of glycosylphosphatidylinositol (GPI), and in mice, mutant red cells can be detected by loss of the GPI-linked protein CD24 (the ‘PNH phenotype’). XK encodes a red cell membrane protein that is covalently linked to the Kell protein, and mutants do not express Kell (the McLeod phenotype). In order to identify McLeod-like cells in mice, we have immunized a Kel -/- mouse and derived a hybridoma (MIMA87) that produces an antibody recognizing the mouse Kell protein. Here we have analyzed red cells collected by tail pricks of C57BL/6 mice. To identify PNH cells, cells were stained with a mixture of anti CD24-PE and Ter119-FITC. For identification of McLeod-like cells, cells were stained with MIMA87 supernatant, then a secondary rabbit anti mouse PE conjugate, and then Ter119-FITC. PNH cells were defined as having <4% of the PE fluorescence of the gated CD24+Ter119+ cells; McLeod-like cells were defined as having <10% of the PE fluorescence of the gated Kel+Ter119+ cells. Prior to treatment, we found that the frequency of McLeod-like red cells in 24 young mice ranged from 0 to 8.5 x 10−6 (mean 1.8 x 10−6). The frequency of PNH red cells in 15 untreated young mice ranged from 0 to 4.2 x 10−6 (mean 1.5 x 10−6). These results are similar to those from human studies and studies in mice with the Hprt gene using lymphocytes. As expected, the frequency of PNH cells increased after an intraperitoneal injection of ethylnitrosourea (ENU). One month post injection, the frequency of PNH cells ranged from 646 to 751 x 10−6 in 3 mice given 100mg/kg and ranged from 1146 to 3602 x 10−6 in 3 animals given 200mg/kg. Surprisingly, there was a much smaller increase in the frequency of McLeod-like red cells in the same mice, a mean of 4.4 x 10−6 (95% CI 3.1 to 5.8 x 10−6) in the mice given 100mg/kg and 15.3 x 10−6 (95% CI 8.1 to 22.4 x 10−6) in the mice given 200mg/kg, compared with 3.3 x 10−6 (95% CI 1.3 to 5.3 x 10−6) in the untreated mice. Approximately 6 months after the injection, the pattern persisted: the mean frequency of McLeod-like red cells was 10.8 x 10−6 (95% CI 4.1 to 17.4 x 10−6) in the mice treated with 200mg/kg compared with 3.2 x 10−6 (95% CI 2.6 to 3.8 x 10−6) in the untreated mice. In contrast, at 6 months, the mean frequency of PNH cells was 9.6 x 10−6 in the untreated mice and 2725 x 10−6 in mice treated with 200mg/kg. A similar pattern was seen in a subsequent experiment using mice of the same strain, and also in an experiment using CD1 mice. We conclude that, as with Pig-a, rare red cells with a spontaneously arising Xk-null phenotype do circulate in mice, but the Xk gene is much less sensitive to the effects of induced mutagenesis with ENU. Citation Format: David J. Araten, Gregory Halverson, Leah Zamechek, Ken Csehak, Wieslawa Kosinska, Joseph Guttenplan. Comparison of the Xk and Pig-a genes as markers for mutations in red cells from mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-153. doi:10.1158/1538-7445.AM2013-LB-153