Figure 1
Inducible HSPC depletion. (A) Mouse model for inducible HSPC depletion; a conditional Rosa26-DTA knock-in allele (R26DTA) was crossed into HSC-CreERT mice. In the latter, the TAM-inducible Cre/estrogen receptor fusion protein (CreERT) is expressed in LT-HSCs and to a lower extent in progenitors. Induction of Cre-mediated recombination by TAM triggers expression of DTA in HSPCs and, thereby, their apoptotic cell death. Scl6E5-CreERT-3′enhancer transgenic mice were designated HSC-CreERT throughout the manuscript. (B) HSC-CreERT/R26DTA/DTA mice were induced by oral gavage of 2 × 0.1 mg/g BW TAM. (C) Flow cytometric analysis of BM HSPCs on day 7 after initiation of TAM. One Cre− control (Cre−) and 1 HSC-CreERT+R26DTA/DTA (Cre+) animal representative of 4 mice per group are shown. Mean frequencies ± SD of the gated population within WBMCs are shown. (D) Total numbers of HSCs and progenitors recovered from 2 femora and 2 tibiae of the animals described in panel B and shown in panel C. Mean numbers of controls (displayed ± SD) were set to 1 (dotted line). See supplemental Figure 1A for gating strategies and Figure 1E and supplemental Figure 2A-B for alternative TAM-induction protocols. (E) BM, spleen, and PB of HSC-CreERT/R26DTA/DTA were analyzed 8 days after TAM administration (n = 5, normalization and display of data as in panel D). (F) WBMCs from TAM-induced (5 × 0.3 mg/g BW) HSC-CreERT/R26DTA/DTA were mixed with equal numbers of B6.CD45.1 competitor WBMCs and transplanted into irradiated B6.CD45.1/CD45.2 recipients (n = 5 per genotype). Recipients were monitored for contribution of the R26DTA/DTA donor (Cre+ or Cre−) to PB neutrophils (NEUT, CD11b+Gr-1hi) and lymphoid cells (LYMPH, FSCloSSCloCD11b−Gr-1−) as well as to BM LSK cells (18 weeks after transfer). Means ± SD are shown. Experiment representative of 2 individual replicates. CLP, common lymphocyte progenitor; FSC, forward scatter; GMP, granulocyte/macrophage progenitor; MEP, megakaryocyte/erythrocyte progenitor; ns, not significant; SD, standard deviation; SSC, side scatter.

Inducible HSPC depletion. (A) Mouse model for inducible HSPC depletion; a conditional Rosa26-DTA knock-in allele (R26DTA) was crossed into HSC-CreERT mice. In the latter, the TAM-inducible Cre/estrogen receptor fusion protein (CreERT) is expressed in LT-HSCs and to a lower extent in progenitors. Induction of Cre-mediated recombination by TAM triggers expression of DTA in HSPCs and, thereby, their apoptotic cell death. Scl6E5-CreERT-3′enhancer transgenic mice were designated HSC-CreERT throughout the manuscript. (B) HSC-CreERT/R26DTA/DTA mice were induced by oral gavage of 2 × 0.1 mg/g BW TAM. (C) Flow cytometric analysis of BM HSPCs on day 7 after initiation of TAM. One Cre− control (Cre−) and 1 HSC-CreERT+R26DTA/DTA (Cre+) animal representative of 4 mice per group are shown. Mean frequencies ± SD of the gated population within WBMCs are shown. (D) Total numbers of HSCs and progenitors recovered from 2 femora and 2 tibiae of the animals described in panel B and shown in panel C. Mean numbers of controls (displayed ± SD) were set to 1 (dotted line). See supplemental Figure 1A for gating strategies and Figure 1E and supplemental Figure 2A-B for alternative TAM-induction protocols. (E) BM, spleen, and PB of HSC-CreERT/R26DTA/DTA were analyzed 8 days after TAM administration (n = 5, normalization and display of data as in panel D). (F) WBMCs from TAM-induced (5 × 0.3 mg/g BW) HSC-CreERT/R26DTA/DTA were mixed with equal numbers of B6.CD45.1 competitor WBMCs and transplanted into irradiated B6.CD45.1/CD45.2 recipients (n = 5 per genotype). Recipients were monitored for contribution of the R26DTA/DTA donor (Cre+ or Cre−) to PB neutrophils (NEUT, CD11b+Gr-1hi) and lymphoid cells (LYMPH, FSCloSSCloCD11b−Gr-1−) as well as to BM LSK cells (18 weeks after transfer). Means ± SD are shown. Experiment representative of 2 individual replicates. CLP, common lymphocyte progenitor; FSC, forward scatter; GMP, granulocyte/macrophage progenitor; MEP, megakaryocyte/erythrocyte progenitor; ns, not significant; SD, standard deviation; SSC, side scatter.

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