miR-144/451^eGFP expression in the hematopoietic system. (A) Schematic overview of the miR-144/451 locus before (top) and after (bottom) insertion of the eGFP expression cassette. The upstream GATA-1 cis-regulatory motif (CRM) and the transcriptional start site (TSS) are indicated. The integration site (chromosome 11: 77886538) of the eGFP cassette within the pre-miR-144 (blue) is shown. The black triangle symbolizes the remaining frt site after Flp-mediated excision of the neo^r cassette and the white rectangle indicates the SV40 poly(A) sequence. The synthetic intronic sequence introduced to ensure efficient nuclear processing and export of the transcript and the newly introduced AflII restriction site used for Southern analysis are shown. (B) Small population of GFP^low cells observed in the CMP population. (i) Live Lin⁻, IL-7R⁻, Sca-1⁻, c-kit⁺ gated cells stained for CD34 and CD16/32. The blue gate indicates the CMP subset.¹⁷  (ii) Cells gated as CMP as a function of FSC-A and GFP intensity. (C) MiR-144/451^eGFP expression is strongly induced on erythroid lineage commitment. (i) Live Lin⁻, IL-7R⁻, Sca-1⁻, c-kit⁺, CD41⁻ gated cells stained for CD150 and CD105. The blue gates indicate granulo-monocyte precursor (GMP) and pre-granulo-monocyte precursor (Pre-GM), pre-megakaryocyte-erythroid precursor (Pre-MegE), Pre-colony forming-unit erythroid precursor (Pre-CFU-E), colony forming unit erythroid (CFU-E), respectively.¹⁹  (ii) The respective populations identified in panel i plotted as a function of FSC-A and GFP intensity. (D) Megakaryocytic precursors (MkP) do not express the reporter. (i) Live Lin⁻, IL-7R<⁻, Sca-1⁻, c-kit⁺ cells stained for CD150 and CD41. The blue gate indicates the MkP subset.¹⁹  (ii) Cells gated as MkP as a function of FSC-A and GFP. (E) All terminal erythropoietic subpopulations are strongly GFP-positive. (i) Live unfractionated bone marrow cells stained for CD71 and Ter119. The blue gates indicate proerythroblast (Pro), basophilic erythroblast (Baso), poly chromatophilic erythroblast (Poly), and ortho-chromatophilic erythroblast (Ortho) populations, respectively.²⁰  (ii) The respective populations identified in panel i plotted as a function of FSC-A and GFP intensity. (F) Mature platelets in the peripheral blood are GFP-negative. (i) EDTA-anticoagulated blood cells plotted as a function of FSC-A and SSC-A. Note the logarithmic axis. (ii) FSC-A^low, SSC-A^low gated cells stained with CD41 and CD61. The blue gate identifies small mature platelets. (iii) Cells gated as platelets as a function of FSC-A and GFP. (G) Mature erythrocytes in peripheral blood are GFP-positive. (i) EDTA-anticoagulated peripheral blood cells stained for Ter119. The blue gate indicates mature RBCs. (ii) Cells gated as RBCs as a function of FSC-A and GFP. (H) Blood smears of fixed peripheral blood show strong endogenous GFP signal from all RBCs. Fluorescence micrographs and corresponding bright field images are shown for wild-type and miR-144/451^+/eGFP. All FACS plot shown are representative of stained miR-144/451^+/eGFP bone marrow cells, except panels F and G, which are miR-144/451^+/eGFP peripheral blood cell stains. The gates for GFP-positive cells in FSC-A vs GFP plots are defined by taking the respective cell population background fluorescence into account using wild-type littermate control stained cells. The percentages shown in the plots indicate the average GFP-positive fraction of the population in question, based on 3 independent experiments.