Generation of two murine lines with GFP inserted into the Gata2 locus. (A) Strategy of GFP knock-ins into exon IS or exon II of the Gata2 gene. The WT Gata2 gene (w) has two alternative untranslated first exons (IS and IG). The GFP gene with a polyadenylation signal sequence (pA) and Neo cassette were integrated into exon IS (ISĜFP:neo) or 5′ to the translational start site in exon II (GFP:neo). Neo cassettes were removed by Cre/loxP-mediated site-specific recombination (ISĜFP, GFP). (B) BglII plus NotI, BglII digested genomic DNA from each mouse line was hybridized with radiolabeled exon III probe as indicated in A. (C) Fluorescent images of EIS-KI and EII-KI whole-mount embryos at E11.5. Green fluorescence was observed only in the midbrain (mb) of EIS-KI embryos whereas, in contrast, EII-KI embryos expressed GFP in the placenta (pl), mesonephros (me), midbrain (mb), and the sensory organs (n, nose; e, ear). (D) Mononuclear cells recovered from EII-KI and EIS-KI bone marrow were stained with PE-conjugated antibodies for hematopoietic lineage markers and analyzed by flow cytometry. The percentages of each quadrangle are shown.

High-level expression of GFP and GATA-2 mRNA in HSC of E1S-KI mice. (A) Lineage negative (Lin⁻) cells recovered from EIS-KI bone marrow were analyzed by flow cytometry after staining with c-Kit and Sca1 antibodies. Cells were also stained by Hoechst 33342, and blue and red fluorescence were measured (H. Blue and H. Red, respectively) to detect the side population (SP). The percentage of cells in each quadrangle is shown. (B) Cells in each quadrangle of the Lin⁻c-Kit⁺ fraction (Lower Left in A) were compared for levels of GATA-2 mRNA by quantitative RT-PCR (normalized to GAPDH). (C) The morphology of Lin⁻GFP⁺Sca1⁺c-Kit⁺ (G⁺L⁻S⁺K⁺) cells from EIS-KI bone marrow are shown after Wright-Giemsa staining. (Scale bar: 10 μm.) (D) Cell-cycle status of G⁺L⁻S⁺K⁺ and G⁻L⁻S⁺K⁺ cells. GFP⁺ and GFP⁻ cells in Sca1⁺c-Kit⁺ fraction of fixed EIS-KI bone marrow were analyzed for their DNA content by Hoechst 33342 flow cytometry. The percentages of cells in S/G₂/M-phases are shown.

Long-term hematopoietic reconstitution activities of G⁺L⁻S⁺K⁺ and equivalent GFP⁻ cells. (A) The contribution of donor (Ly5.1⁺ EIS-KI mice)-derived myeloid (Mac⁺ and Gr1⁺, Left) and lymphoid (Thy1.2⁺ and B220⁺, Right) cells in lethally irradiated Ly5.2⁺ recipient mice was measured for 8 months after transplantation. Total Lin⁻ cells (2 × 10⁵, dotted lines/triangles), 4 × 10³ Lin⁻GFP⁺ (black circles), or 2 × 10⁵ Lin⁻GFP⁻ (gray boxes) cells were examined. (B) The frequency of long-term (5 months) reconstituting HSC in G⁺L⁻S⁺K⁺ (black circles) and comparable GFP⁻ (gray boxes) fractions was investigated by limiting dilution assay. The gray lines show the 37%-negative points based on a Poisson distribution. (C) Bone marrow cells recovered from primary recipients were divided into GFP⁺ and GFP⁻, and 2 × 10⁴ cells from each population were secondary transplanted. The chimerism of transplanted cells in the peripheral myeloid (Left) and lymphoid (Right) compartments after transplantation is shown.

HSCs are dormant and solitary in the bone marrow niche. (A and B) Frozen bone sections from EIS-KI mice were stained with PE-conjugated anti-Sca1 antibody. The green fluorescent images (Left) were merged with Nomarski and PE images (Right). A and B are photographs from independent mice. GFP or Sca1 single-positive cells are indicated with green (long arrows) or red, respectively. Yellow cells (short arrows) indicate GFP/Sca1 double-positive cells that reside by the edge of the bone marrow. The bone marrow sections were also stained with anti-GFP antibody (arrows, brown) and alkaline phosphatase (purple in C) or Alcian blue (D). GFP⁺ cells are in direct contact with purple-stained osteoblasts (C), but not with the Alcian blue⁺ cartilage (D). (E) Green fluorescent (Left) and bright field (Right) images of living bone marrow from the scapula of an EIS-KI mouse 1 day after 5-FU injection. GFP⁺ cells are detected near the edge of the bone marrow. (F and G). Two days after 5-FU administration, GFP⁺ cells are apparently quiescent, deep in the bone marrow. Time-lapse images of E, F, and G are exhibited in Movies 1–3. The movies demonstrate that GFP⁺ cells are almost completely immobilized in the bone marrow, whereas the surrounding GFP⁻ cells move vigorously.