Escape from Notch suppression by heterozygous DNMAML-GFP occurs frequently during T-ALL development. (A) Survival curve showing the fraction of LC (N = 5 mice), LCR (N = 15), LCDR (N = 11), and LCD (N = 6) mice developing T-ALL over time. (B) Flow cytometric analysis of peripheral blood leukocytes showing expression of DNMAML-GFP of a representative LCDR mouse. Both DNMAML-GFP⁺ and DNMAML-GFP⁻ (internal negative control) subsets were analyzed for immature T-cell blasts using CD4 and CD8 markers at 6 weeks, 22 weeks, and 24 weeks of age. (C) Flow cytometric analysis of thymic lymphomas of 3 representative LCDR mice with T-ALL showing expression of DNMAML-GFP, CD4, and CD8. (D) DNA sequence analysis of endogenous Notch1 in thymic lymphomas of 7 LCDR mice with T-ALL. Mutations in the HD and PEST domains are shown in addition to the mutational consequence in the protein. Mutations were detected upon morbidity at 110 days (total average) of age, 107 days (LCDR1), 105 days (LCDR2), 80 days (LCDR3), 167 days (LCDR4), and 90 days (LCDR6). (E) Genomic PCR strategy to determine the presence of DNMAML-GFP at the ROSA26 locus. The DNMAML-GFP (“DNMAML”) allele generates a ∼400-bp fragment whereas the WT allele generates a ∼700-bp fragment. (F) Genomic PCR analysis of 3 thymic lymphomas for the DNMAML-GFP and WT alleles at the ROSA26 locus. cDNA, complementary DNA; ID, identification; SA, splice acceptor.