NrasG12D/G12D,C181Smice develop fatal hematologic disease characterized by S181C revertant mutations. (A) Survival of NrasG12D/G12D,C181S mice (n = 24) superimposed on that of the NrasG12D mice shown in Figure 1G. (B-C) Spleen weights (B; n = 6) and WBC counts (C; n = 5) in diseased NrasG12D/G12D,C181S mice compared with WT controls (see supplemental Figure 3). (D-F) Immunophenotypic characterization of red blood cell-lysed blood (D), BM (E), and spleen cells (F) by flow cytometry. (G) In the top panel, schematics indicating the 2 sets of primers used to amplify and sequence either both Nras alleles or the C181S-engineered allele. The bottom panel shows representative data from a diseased mouse with both reduced levels of the G>C (C181S) mutation in BM DNA and preservation of the FRT sequence. (H) Allelic frequency of the FRT sequence and the C181S mutation in the tail DNA and BM DNA amplified with primers indicated in panel G. Each symbol corresponds to an individual mouse. Bars are average ± SEM. *P < .05; **P < .1. ns, not significant.