American Society of Hematology

Figure 2

$Figure 2. Acute ablation of Hif-2α has no impact on survival and function of HSCs (A) Experimental design. BM cells from untreated Hif-2αfl/fl;Mx1-Cre mice and control mice (without Mx1-Cre) were mixed with CD45.1+ WT competitor BM and transplanted into lethally irradiated recipients. After confirmation of equal multilineage reconstitution 8 weeks after transplantation, the mice were treated with 6 doses of pIpC and were analyzed 2, 4, and 36 weeks after the last pIpC administration. (B) Representative gel showing PCR amplification of genomic DNA from donor-derived CD45.2+ fraction of the PB from the pIpC-treated recipient mice shown. The analysis was performed 2 weeks after the last dose of pIpC. (C) The graphs show the percentage of CD45.2+ donor-derived cells measured in total BM, BM LSK, and HSC compartments of the recipients. Data are mean ± SEM, n = 7 per group per time point. The data are representative of 2 independent experiments. (D) Total numbers of HSCs and primitive progenitor cell populations from 10- to 12-week-old Hif-1αfl/fl;Hif-2αfl/fl;Vav-iCre mice (n = 6) and control mice (Vav-iCre–negative; n = 5). Data are mean ± SEM. (E) Total numbers (per 2 femurs and 2 tibias) of BM B-lymphoid (CD19+B220+), myeloid (CD11b+Gr1+), and erythroid (Ter119+) cells from Hif-1αfl/fl;Hif-2αfl/fl;Vav-iCre mice (n = 4) and control mice (n = 6). Data are mean ± SEM. (F) Experimental design. Lethally irradiated recipients were transplanted with 500 000 CD45.2+ BM mononuclear cells from Hif-1αfl/fl;Hif-2αfl/fl;Vav-iCre mice or control mice (Hif-1αfl/fl;Hif-2αfl/fl without Vav-iCre) together with 500 000 BM mononuclear cells from WT CD45.1+ mice. Gene deletion was confirmed 16 weeks after transplantation (G). Chimerism analysis was performed 3, 7, 12, and 16 weeks after transplantation (H; supplemental Figure 4). (G) Representative gel showing PCR amplification of genomic DNA from CD45.2+ fraction of the BM from recipient mice transplanted with Hif-1αfl/fl;Hif-2αfl/fl;Vav-iCre cells and control cells. Samples were taken 16 weeks after transplantation. (H) PB chimerism in recipients of control cells and Hif-1α/Hif-2α–deficient BM cells. Data are mean ± SEM (n = 3-5 per group). Representative data of 3 independent transplantation experiments are shown. Δ, excised allele; fl, undeleted conditional allele; WT, wild-type allele.$

Acute ablation of Hif-2α has no impact on survival and function of HSCs (A) Experimental design. BM cells from untreated Hif-2α^fl/fl;Mx1-Cre mice and control mice (without Mx1-Cre) were mixed with CD45.1⁺ WT competitor BM and transplanted into lethally irradiated recipients. After confirmation of equal multilineage reconstitution 8 weeks after transplantation, the mice were treated with 6 doses of pIpC and were analyzed 2, 4, and 36 weeks after the last pIpC administration. (B) Representative gel showing PCR amplification of genomic DNA from donor-derived CD45.2⁺ fraction of the PB from the pIpC-treated recipient mice shown. The analysis was performed 2 weeks after the last dose of pIpC. (C) The graphs show the percentage of CD45.2⁺ donor-derived cells measured in total BM, BM LSK, and HSC compartments of the recipients. Data are mean ± SEM, n = 7 per group per time point. The data are representative of 2 independent experiments. (D) Total numbers of HSCs and primitive progenitor cell populations from 10- to 12-week-old Hif-1α^fl/fl;Hif-2α^fl/fl;Vav-iCre mice (n = 6) and control mice (Vav-iCre–negative; n = 5). Data are mean ± SEM. (E) Total numbers (per 2 femurs and 2 tibias) of BM B-lymphoid (CD19⁺B220⁺), myeloid (CD11b⁺Gr1⁺), and erythroid (Ter119⁺) cells from Hif-1α^fl/fl;Hif-2α^fl/fl;Vav-iCre mice (n = 4) and control mice (n = 6). Data are mean ± SEM. (F) Experimental design. Lethally irradiated recipients were transplanted with 500 000 CD45.2⁺ BM mononuclear cells from Hif-1α^fl/fl;Hif-2α^fl/fl;Vav-iCre mice or control mice (Hif-1α^fl/fl;Hif-2α^fl/fl without Vav-iCre) together with 500 000 BM mononuclear cells from WT CD45.1⁺ mice. Gene deletion was confirmed 16 weeks after transplantation (G). Chimerism analysis was performed 3, 7, 12, and 16 weeks after transplantation (H; supplemental Figure 4). (G) Representative gel showing PCR amplification of genomic DNA from CD45.2⁺ fraction of the BM from recipient mice transplanted with Hif-1α^fl/fl;Hif-2α^fl/fl;Vav-iCre cells and control cells. Samples were taken 16 weeks after transplantation. (H) PB chimerism in recipients of control cells and Hif-1α/Hif-2α–deficient BM cells. Data are mean ± SEM (n = 3-5 per group). Representative data of 3 independent transplantation experiments are shown. Δ, excised allele; fl, undeleted conditional allele; WT, wild-type allele.

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