Analyses of Tet gene expression and 5-hmC levels in BM cells of WT, Tet2^+/−, and Tet2^−/− mice. (A) Analysis of the mRNA expression levels of each Tet genes in BM cells of 6- to 7-week-old WT (n = 4), Tet2^+/− (n = 6) and Tet2^−/− (n = 6) mice by quantitative real-time PCR. The relative mRNA expression of Tet2, Tet1, and Tet3 was determined using ß-actin as internal calibrator. The mRNA expression levels are reported as relative expression units to the respective Tet expression in WT mice. (B-D) Genomic DNA was extracted from BM cells of 6- to 7-week-old WT (n = 3), Tet2^+/− (n = 4), and Tet2^−/− (n = 4) mice and blotted onto nitrocellulose membrane after 2-fold serial dilution. 5-hmC (B) and 5-mC (C) levels were detected with an anti–5-hmC (ActiveMotif, #39791) or 5-mC (Calbiochem; NA#81) antibody. Methylene blue staining was performed to ensure equal spotting of total DNA on the membranes. Quantification of the signal density (D) was shown. *P < .05, **P < .01 ***P < .001, ****P < .0001 (E-H) Tet2^−/− mice developed a phenotype resembling characteristics of CMML as early as 2-4 months of age. WT (n = 18), Tet2^+/− (n = 24), and Tet2^−/− (n = 28) mice were killed at 2-4 months of age and were analyzed for PB WBC (E), monocyte (MO, F), and RBC (G) counts. (H) Representative May-Giemsa stained PB smears prepared from WT, Tet2^+/−, and Tet2^−/− mice at 3-4 months of age. Black arrows indicate monocytes, and red arrows, indicate neutrophils.