Figure 3

CALR mutant mice develop ET. (A) A transgenic construct was generated by introducing human CALRdel52 mutant cDNA into the pSP65-H2K-i-LTR vector. (B) The level of expression of human CALRdel52 mRNA in different lineage cells. Left: the average expression level of CALR mRNA in two human megakaryocytic leukemia cell lines (CHRF288 and CMK11-5) is set at 1. The level of expression of human CALRdel52 mRNA in BM cells from CALRdel52-TG is 0.64-fold smaller than the average expression of CALR mRNA in three human megakaryocytic cell lines. Right: the expression level of CALRdel52 in whole BM cells is set at 1. Compared with the expression of CALRdel52 in BM cells, that in LSKs, erythromegakaryocyte progenitors (MEP) and CD41+ BM cells is higher, whereas that in common myeloid progenitor (CMP), granulocyte–macrophage progenitor (GMP) and Mac1/Gra1+ BM cells is lower. (C) The average complete blood cell count in CALRdel52-TG mice every 8 weeks after birth (n=30). Compared with WT mice (n=29), TG mice show thrombocytosis at 8 weeks and later. There is no difference in WBC count or Hb level between WT and TG mice. **P<0.01 vs WT mice. (D) Liver and spleen weights from 9- to 13-week-old mice. CALRdel52-TG mice (n=10) do not show hepatosplenomegaly compared with WT mice (n=9). (E) Histological analysis of 24-week-old CALRdel52-TG mice. BM and spleen stained with hematoxylin and eosin (HE) (a, c, d and f); BM with Gomori silver stain (b and e). CALRdel52-TG mice show increased numbers of megakaryocytes in BM and spleen (d and f). There is no fibrosis in BM (e). Lower left panel: the nucleated cells in one femur and one tibia from TG (n=10) or WT mice (n=9) were counted. There is no difference in cellularity between the two groups. Lower center panel: the number of megakaryocytes per 10 HPF in BM was higher in TG (n=5) than in WT mice (n=5). Lower right panel: sizes of megakaryocytes in BM. In each mouse, the diameters of 100 megakaryocytes were measured. Megakaryocytes in TG mice (n=5) are significantly larger than those in WT mice (n=5). P-values are shown. (F) Left panel: electron microscopic appearance of mature megakaryocytes found in WT and TG BM (magnification × 1000 in upper, × 4000 in lower). α-Granules and nuclear lobes are well developed in megakaryocytes in TG. Right panel: the numbers of α-granules were counted in 50 randomly selected megakaryocytes from each of three WT mice and three TG mice. P-values are shown. (G) Kaplan–Meier plot of WT and TG mice. (H) 5 × 10⁶ BM cells from either TG or WT mice together with 2 × 10⁵ WT BM cells were transplanted into irradiated WT mice. Recipient mice transplanted with TG BM cells (n=12) exhibit thrombocytosis, but like recipients transplanted with WT BM cells they do not show erythrocytosis or leukocytosis (n=12). *P<0.05 and **P<0.01 vs WT mice. All data are presented as means±s.e.m. To assess the statistical significance between the two groups, the two-tailed Student’s t-test was used (D–F). For the comparison of the hematological values between CALRdel52-TG and WT mice, analysis of variance with repeated measures was used (C and H). Statistical analyses of the survival were performed with the log-rank test (G).