Figure 1.
Figure 1. Competitive transplantations with JAK2-V617F (V617F) and WT BM cells. (A–D) Transplantation experiments in which GFP is expressed in the WT competitor cells, allowing indirect monitoring of the mutant allele burden. The experiment was performed twice, total n = 12 mice per group, one experiment with n = 7 is shown. (A) Schematic drawing showing the design of the experiment. The time course of PB parameters and chimerism were determined for the erythroid (TER119), platelet (CD61), granulocytic (Gr1), and B cell (B220) lineages. (B) Spleen weight at terminal workup at 40 wk (n = 7 per group). (C) Flow cytometry scattergrams showing the LSK gating. The bar graphs show the percentages of LSKs in lineage-negative cells in the BM and the chimerism within the LSK population (n = 5 per group). (D) Histopathology taken at 40 wk after transplantation (one representative mouse per group is shown). Bars, 50 µm. (E–H) Transplantation experiments in which V617F and GFP is coexpressed in the same cells, allowing direct monitoring of the mutant allele burden. The experiment was performed twice, total n = 10 of which one experiment is shown (n = 5). (E) Schematic drawing of the experimental design and results of blood counts and chimerism are shown. (F) Spleen weight at terminal workup at 41 wk (n = 5 for WT and n = 2 for V617F). (G) Gating strategy for the quantification of myeloid progenitors and HSCs. (H) Flow cytometry quantification of progenitor and stem cell populations in BM and spleen at 41 wk after transplantation (n = 5 for WT and n = 2 for V617F). (I–K) Transplantation of BM from experiment A into secondary recipients (n = 5 recipients per group, the experiment was performed once). (I) Blood counts and chimerism are shown. (J) Spleen weight of secondary recipients at 44 wk (n = 5 per group). (K) Gating and quantification of LSK cells and chimerism within the LSK population (n = 5 per group). (L–N) Transplantation of BM from experiment I into tertiary recipients (n = 5 recipients per group, the experiment was performed once). (L) Blood counts and chimerism are shown. The dashed line represents one mouse that developed a marked neutrophilia. (M) Spleen weight of tertiary recipients taken at 32 wk after transplantation (n = 3). (N) Gating and quantification of LSK cells and chimerism within the LSK population (for quantification n = 3). Statistical analysis was conducted using the Student’s t test or one-way ANOVA with Bonferroni’s post-hoc multiple comparison test. Error bars represent ±SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001. n.a., not available.

Competitive transplantations with JAK2-V617F (V617F) and WT BM cells. (A–D) Transplantation experiments in which GFP is expressed in the WT competitor cells, allowing indirect monitoring of the mutant allele burden. The experiment was performed twice, total n = 12 mice per group, one experiment with n = 7 is shown. (A) Schematic drawing showing the design of the experiment. The time course of PB parameters and chimerism were determined for the erythroid (TER119), platelet (CD61), granulocytic (Gr1), and B cell (B220) lineages. (B) Spleen weight at terminal workup at 40 wk (n = 7 per group). (C) Flow cytometry scattergrams showing the LSK gating. The bar graphs show the percentages of LSKs in lineage-negative cells in the BM and the chimerism within the LSK population (n = 5 per group). (D) Histopathology taken at 40 wk after transplantation (one representative mouse per group is shown). Bars, 50 µm. (E–H) Transplantation experiments in which V617F and GFP is coexpressed in the same cells, allowing direct monitoring of the mutant allele burden. The experiment was performed twice, total n = 10 of which one experiment is shown (n = 5). (E) Schematic drawing of the experimental design and results of blood counts and chimerism are shown. (F) Spleen weight at terminal workup at 41 wk (n = 5 for WT and n = 2 for V617F). (G) Gating strategy for the quantification of myeloid progenitors and HSCs. (H) Flow cytometry quantification of progenitor and stem cell populations in BM and spleen at 41 wk after transplantation (n = 5 for WT and n = 2 for V617F). (I–K) Transplantation of BM from experiment A into secondary recipients (n = 5 recipients per group, the experiment was performed once). (I) Blood counts and chimerism are shown. (J) Spleen weight of secondary recipients at 44 wk (n = 5 per group). (K) Gating and quantification of LSK cells and chimerism within the LSK population (n = 5 per group). (L–N) Transplantation of BM from experiment I into tertiary recipients (n = 5 recipients per group, the experiment was performed once). (L) Blood counts and chimerism are shown. The dashed line represents one mouse that developed a marked neutrophilia. (M) Spleen weight of tertiary recipients taken at 32 wk after transplantation (n = 3). (N) Gating and quantification of LSK cells and chimerism within the LSK population (for quantification n = 3). Statistical analysis was conducted using the Student’s t test or one-way ANOVA with Bonferroni’s post-hoc multiple comparison test. Error bars represent ±SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001. n.a., not available.

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