The presence of GFI136N affects the GMP bone marrow fraction in Gfi1^36N/36N knock-in mice. (A) Representative flow cytometric analysis of CMPs, GMPs, and MEPs of the indicated mouse strains. Numbers indicate percentage of the different populations with regard to the total bone marrow cells. (B) Total number of GMPs in the different mouse strains (both hind limbs; n = 24 for Gfi1^+/+, n = 15 for Gfi1^−/−, n = 15 for Gfi1^36N/36N, n = 7 for Gfi1^36S/36S, n = 4 for Gfi1^36N/+, n = 3 for Gfi1^36N/+). *P ≤ .05. (C) Cell cycle progression of the GMPs from the indicated strains (n = 5 for Gfi1^+/+, n = 3 for Gfi1^−/−, n = 5 for Gfi1^36N/36N, n = 5 for Gfi1^36S/36S,, n = 5 for Gfi1^36N/+, n = 3 for Gfi1^36N/+). *P ≤ .05. (D) Heat map representing the different mRNA expression profiles in GMPs from the indicated mouse strains. Genes expressed in GMPs at more than a 2-fold difference in the comparisons, Gfi1^−/− vs Gfi1^+/+, Gfi1^36N/36N vs Gfi1^36S/36S, or Gfi1^36N/36N vs Gfi1^+/+, were analyzed by hierarchical clustering using Wards method (similarity measure: half square eucledian distance). (E-F) Gene set enrichment analysis showed that sets of genes differentially expressed in mature adult hematopoietic cells compared with stem cells (HSC_Mature_Adult) and in MLL-AF9 leukemia (Kumar_HoxA_Diff) are enriched in the list of genes that are differentially expressed between Gfi1^36S/36S and Gfi1^36N/36N GMPs. (G) Table of results of KEGG pathway analysis showing that genes differentially expressed between Gfi1^36S/36S and Gfi1^36N/36N GMPs fall into the indicated pathways, the one composing hematopoietic cell lineage genes being the most significant. (H) Venn diagram comparing the number of overlapping genes differentially expressed in GMPs from the indicated mouse strains. (I) A total of 450 GMPs from Gfi1^36S/36S and Gfi1^36N/36N mice were sorted directly on methylcellulose, and 20 000 cells were replated from the emerging colonies every 10 days (see also “In vitro colony assay”). *P = .05. n = 3 for all genotypes. (J) Schematic outline of transplantation experiment. A total of 2500 GMPs were sorted from Gfi1^36S/36S or Gfi1^36N/36N mice and transplanted together with 10⁵ bone marrow cells from CD45.1⁺ mice into lethally irradiated CD45.1⁺ mice. (K) At 24 days after transplantation, the number of CD45.1⁺ (all wt) and CD45.2⁺ (either Gfi1^36S/36S or Gfi1^36N/36N) GMPs was determined. (L-M) At 24 days after transplantation of either Gfi1^36S/36S or Gfi1^36N/36N CD45.2⁺ GMPs, the percentage of CD45.2⁺ GMPs was determined in the recipient mice. Whereas no CD45.2⁺, Gfi1^36S/36S GMPs were detectable, ∼ 1% of the GMPs were CD45.2⁺Gfi1^36N/36N GMPs.