Figure 2
Figure 2. Analysis of JAK2-Ex12 transgene copy number and transgene expression. (A) Schematic drawing of Cre-mediated rearrangements of the JAK2-Ex12 transgene. The region of the transgene containing the mutated JAK2 exon 12 is shown enlarged and the DNA connecting the 2 copies of the transgene was omitted (inclined double lines connected by dashed line). The head-to-tail orientation of the 2 copies of the transgene was confirmed by PCR with primers flanking the ends of the construct. The inactive transgene configuration (top); Cre recombination of adjacent loxP sites (middle) leads to reversal of the orientation and activation of 2 copies of the transgene (N542-E543del); and Cre recombination of distant loxP sites that are in parallel orientation results in the excision of 1 copy of the transgene (bottom). (B) The Ex12 transgene copy number was determined by quantitative PCR in white blood cells (WBCs), and in sorted Gr1+, Mac1+, CD3+, and B220+ cells from peripheral blood (PB) of MxCre;Ex12 mice 24 weeks after pIpC injection (n = 6 mice per group). (C) Ratio of human JAK2-N542-E543del to mouse Jak2 mRNA expression determined by RT-PCR in BM and peripheral blood. Results from total BM cells or sorted erythroid (CD71+) and myeloid (Gr1+) cells, as well as platelets, granulocytes (Gr1+), monocytes (Mac1+), T cells (CD3+), and B cells (B220+) from peripheral blood of MxCre;Ex12 mice 24 weeks after pIpC induction are shown. Boxes represent the interquartile range that contains 50% of the values and the whiskers indicating the range containing 95% of the values. Horizontal lines indicate the mean values (n = 6 mice per group). (D) Correlation of hemoglobin levels with the ratio between human mutated JAK2/mouse wild-type (WT) Jak2 mRNA expression in BM of MxCre;Ex12 (red dots, n = 7) and MxCre;V617F mice (blue dots, n = 6), 24 weeks after pIpC induction. (E) Immunoblot analysis of Jak2 protein expression in BM cell lysates. The upper panel shows the quantification of western blots probed with an antibody that preferentially recognizes human Jak2 protein (Imgenex). The intensities of the bands in the lower panel were quantified and differences in loading were normalized using β-actin antibodies. The values are shown as the fold change of Jak2 in WT mice. For comparison, the blot was reprobed with a Jak2 antibody (Millipore) that does not discriminate between human and mouse Jak2 proteins. WT (black), MxCre;Ex12 (red), and MxCre;V617F (blue) mice (n = 2 per group). One-way ANOVA with subsequent Bonferroni post-test was used. *P < .05.

Analysis of JAK2-Ex12 transgene copy number and transgene expression. (A) Schematic drawing of Cre-mediated rearrangements of the JAK2-Ex12 transgene. The region of the transgene containing the mutated JAK2 exon 12 is shown enlarged and the DNA connecting the 2 copies of the transgene was omitted (inclined double lines connected by dashed line). The head-to-tail orientation of the 2 copies of the transgene was confirmed by PCR with primers flanking the ends of the construct. The inactive transgene configuration (top); Cre recombination of adjacent loxP sites (middle) leads to reversal of the orientation and activation of 2 copies of the transgene (N542-E543del); and Cre recombination of distant loxP sites that are in parallel orientation results in the excision of 1 copy of the transgene (bottom). (B) The Ex12 transgene copy number was determined by quantitative PCR in white blood cells (WBCs), and in sorted Gr1+, Mac1+, CD3+, and B220+ cells from peripheral blood (PB) of MxCre;Ex12 mice 24 weeks after pIpC injection (n = 6 mice per group). (C) Ratio of human JAK2-N542-E543del to mouse Jak2 mRNA expression determined by RT-PCR in BM and peripheral blood. Results from total BM cells or sorted erythroid (CD71+) and myeloid (Gr1+) cells, as well as platelets, granulocytes (Gr1+), monocytes (Mac1+), T cells (CD3+), and B cells (B220+) from peripheral blood of MxCre;Ex12 mice 24 weeks after pIpC induction are shown. Boxes represent the interquartile range that contains 50% of the values and the whiskers indicating the range containing 95% of the values. Horizontal lines indicate the mean values (n = 6 mice per group). (D) Correlation of hemoglobin levels with the ratio between human mutated JAK2/mouse wild-type (WT) Jak2 mRNA expression in BM of MxCre;Ex12 (red dots, n = 7) and MxCre;V617F mice (blue dots, n = 6), 24 weeks after pIpC induction. (E) Immunoblot analysis of Jak2 protein expression in BM cell lysates. The upper panel shows the quantification of western blots probed with an antibody that preferentially recognizes human Jak2 protein (Imgenex). The intensities of the bands in the lower panel were quantified and differences in loading were normalized using β-actin antibodies. The values are shown as the fold change of Jak2 in WT mice. For comparison, the blot was reprobed with a Jak2 antibody (Millipore) that does not discriminate between human and mouse Jak2 proteins. WT (black), MxCre;Ex12 (red), and MxCre;V617F (blue) mice (n = 2 per group). One-way ANOVA with subsequent Bonferroni post-test was used. *P < .05.

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