Figure 1.
CD11c-Cre mice have minimal GFP+ DCs in spleen.
Flow cytometric analysis of CD11c-Cre mouse spleen cells for their GFP expression in A) DCs, myeloid cells, T- and B-lymphocytes and natural killer (NK) cells. B) Flow cytometric analysis of the same mouse spleen cells for GFP expression in different DC subsets including pDCs (CD11c+B220+CD8−CD11b−Ly6C+), CD8+ DCs (CD11c+CD8+CD11b−MHCII+), CD8−CD11b+ DCs (CD11c+CD8−CD11b+MHCII+), inactivated (CD11c+MHCII+CD8+) and activated DCs (CD11c+MHCII+CD8−) and DC progenitors (Lineage−CD11c+CD11b−B220−CD43+). C) Analysis of CD11b+ spleen cells for GFP expression during their maturation from group I (CD11c−Ly6Chi), group II (CD11c−Ly6Clow), to group III (CD11c−Ly6C−), and group IV (CD11c+Ly6C−).
Figure 2.
Generation of mice bearing both CD11c-Cre and Rosa26 loxP-STOP-loxP YFP.
Diagrams show A) the construct of loxP (triangles) flanked transcription stop signal sequence (SA) followed by YFP under the control of Rosa26 locus; and B) the 5.3 kb CD11c proximal promoter controlled Cre recombinase followed by IRES and GFP. C) the diagram demonstrates the breeding strategy of generating mice carrying both CD11c-Cre and Rosa26 loxP-STOP-loxP. D) PCR on genomic DNA from offspring mice of the breeding pairs in C. Mice carrying both CD11c-Cre and Rosa26 loxP-STOP-loxP demonstrate both the 420 bp Cre product and the 600 bp YFP product.
Figure 3.
CD11c-Cre mediated recombination to activate YFP expression in spleen cells.
Flow cytometric analysis of spleen cells from mice bearing both CD11c-Cre and Rosa26 loxP-STOP-loxP for the Cre activated YFP expression in A) various lineages including CD11c+ DCs, Gr1+ myeloid cells, B220+ B- and CD3+ T-lymphocytes, and in B) DC subsets including CD8+ DCs, CD8−CD11b+ DCs, pDCs, inactivated and activated DCs. C) Analysis of YFP expression in CD11b+ DCs during their maturation from group I to group IV.
Figure 4.
CD11c-Cre mediated recombination to activate YFP expression in mouse bone marrow hematopoietic stem and progenitor cells.
Flow cytometric analysis of bone marrow cells from mice bearing both CD11c-Cre and Rosa26 loxP-STOP-loxP for YFP expression in A) lineage negative cells, early myeloid progenitors (LK), stem cells (LSK) and CMPs, GMPs, MEPs; and in B) DC progenitor cells including MDPs and CDPs.
Figure 5.
CD11c-Cre mediated recombination to activate YFP expression in mouse bone marrow cells cultured with GM-CSF or FLT3L.
Flow cytometric analysis of bone marrow cells from mice bearing both CD11c-Cre and Rosa26 loxP-STOP-loxP for their YFP expression in CD11c+ and CD11c− cells after cultured with GM-CSF or FLT3L for 6 days A) and C), and for 11 days B) and D), respectively.
Figure 6.
CD11c-Cre mediated recombination to activate YFP expression in spleen cells after GM-CSF administration in mice.
Mice bearing both CD11c−Cre and Rosa26 loxP-STOP-loxP were injected with GM-CSF followed by flow cytometric analysis of their spleen cells for YFP expression in A) different lineages including DCs, myeloid cells, B- and T-lymphocytes, and in B) DC subsets including CD8+ DCs, CD8−CD11b+ DCs and pDCs. C) Analysis of YFP expression in CD11b+ DCs during their maturation from group I to group IV.
Figure 7.
CD11c proximal promoter contains binding sites for PU.1 and IRF-4, which are essential for GM-CSF or FLT3L-induced CD11c activation.
A) Diagram demonstrating the human 5.3 kb CD11c proximal promoter. Transcription factors binding to this region according to ChIP-seq analysis of existing data are illustrated in the diagram. B) ChIP assays on cultured mouse bone marrow cells with GM-CSF for the binding of Pu.1 and Irf-4 to the CD11c proximal promoter region. C) Quantitative PCR on reverse transcribed mRNA from mouse bone marrow cells cultured with GM-CSF or FLT3L to evaluate expression of Pu.1 and Irf-4. Bone marrow cells from mice bearing both CD11c-Cre and Rosa26 loxP-STOP-loxP were transduced with retrovirus carrying either scrambled control shRNA or shRNA against Pu.1 followed by puromycin selection of transduced cells. Immunoblotting D) and Flow cytometric analysis of the transduced cells for the expression YFP in both CD11c+ and CD11c− cells 11 days after culturing with GM-CSF E), or FLT3L F).