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Blood, Vol. 111, Issue 8, 3931-3940, April 15, 2008
Ratio of mutant JAK2-V617F to wild-type Jak2 determines the MPD phenotypes in transgenic mice
Blood Tiedt et al.
111: 3931
Supplemental materials for: Tiedt et al
Files in this Data Supplement:
- Table S1. Blood counts (PDF, 1.82 MB) -
Blood counts were determined on a Advia 120 Hematology Analyzer using the “Multispecies Software” with the settings for mouse blood. Top (green bar): blood counts of VavCre;FF1/+ hemizygous mice (n=6). The mean values at age of 12 weeks are given as indicated. Row labeled SD: standard deviation for each of the parameters. Row labeled P value: Student t-test for each of the values obtained for the VavCre;FF1 mice compared to the corresponding values for control mice. First orange bar: blood counts of MxCre;FF1/+ hemizygous mice that received 6 consecutive injections of pIpC (n=3). Blood was drawn 12 weeks after the pIpC injections. Annotations as above. Note that hemoglobin is lower than in MxCre;FF1 mice that received 3 injections of pIpC. Second orange bar: blood counts of MxCre;FF1/+ hemizygous mice that received 3 injections of pIpC (n=11). Blood was drawn 11 weeks after the pIpC injections. Third orange bar: blood counts of MxCre;FF1/+ hemizygous mice that received a single injection of pIpC (n=5). Blood was drawn 10-12 weeks after the pIpC injections. Note that the hemoglobin concentration is higher than in MxCre;FF1 mice that received 3 injections of pIpC. Fourth orange bar: blood counts of MxCre;FF1/FF1 homozygous mice that received 3 consecutive injections of pIpC (n=2). Blood was drawn 10-12 weeks after the pIpC injections. Note that these mice showed the highest hemoglobin values. However, only two mice homozygous for FF1 were available for analysis. Blue bar: blood counts of the VF1 transgenic strain (n=23). Gray bar (bottom): blood counts of single transgenic control mice (MxCre or FF1/+) that received 3 consecutive injections of pIpC (n=19). Blood was drawn 12 weeks after the pIpC injections.
- Figure S1. Flow cytometric analysis of hematopoietic lineages in bone marrow and spleen (JPG, 104 KB)
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(A) Analysis of erythroid cells by staining for transferrin receptor (CD71) and Ter119. Note the decrease of erythropoiesis in bone marrow and increase in the spleens of both VavCre;FF1 and MxCre;FF1 mice. (B) Myeloid cells were stained with antibodies against Ly-6G (Gr1) and integrin  M (Mac1). Both transgenic mice showed expansion of myeloid (Gr1+/Mac1+) cells in bone marrow and spleen. (C) T cells were stained with CD3 and B cells with B220. A reduction in the percentage of B cells in the bone marrow and spleen and of T cells in spleen was noted. (D) Megakaryocytes were stained with antibodies against glycoprotein IIb/IIIa (CD41 and CD61). A strong increase in CD41/CD61-double positive cells was noted in the bone marrow and spleen of both VavCre;FF1 and MxCre;FF1 mice. This is in good agreement with the histological findings (Figure 3).
- Figure S2. Analysis of active transgene copy numbers in VavCre;FF1 mice and MxCre;FF1 mice after different numbers of pIpC injections (JPG, 32 KB)
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- Figure S3. Analysis of JAK2 expression in MPD patients and normal controls (JPG, 54 KB)
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 CT values for JAK2 expression were calculated using three different genes for normalization: ribosomal protein L19 (RPL19), beta-2-microglobulin (B2M) and beta-glucuronidase (GUSB). Relative expression values were then calculated by the CT method using one healthy control as calibrator. Significances were evaluated by pairwise Mann-Whitney tests, P<0.05 was marked with asterisks. Even though a trend towards higher JAK2 expression in PV and PMF patients was observed in each case, the results show that the choice of normalization gene has a strong impact on the amplitude of gene expression differences.
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