Test of constructs. (A) Schematic design. An XhoI linkered fragment encoding the intracellular domain of murine c-mpl was PCR-amplified by using Pfu polymerase and the following primer pairs: 5′-GGCTCGAGAAGTGGCAATTTCCTGCG-3′ and 5′-GGCTCGAGGGGCTGCTGCCAATAGC-3′. After sequence confirmation, the XhoI-digested fragment was inserted into the SalI site of the construct F3 (5) to produce F3mpl. F3mpl contains a myristylation domain to direct localization to the inner surface of the cell membrane, three copies of the FK506-binding peptide FKBP12, the intracellular portion of c-mpl, and a hemagglutinin epitope tag to permit detection of the fusion protein by Western blot assay. F1mpl differs from F3mpl in that it contains only a single FKBP12 site. These constructs were used to generate Ba/F3 clones expressing high levels of the fusion protein as described previously (5). (B) Cell proliferation (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, MTT) assays for transfected Ba/F3 cell clones expressing either F3mpl (solid symbols) or F1mpl (open circles) were performed as described previously (5). Peak proliferative responses were observed at a concentration of 100 nM FK1012. (C) MTT assays for retrovirally transduced Ba/F3 cell clones expressing F1mpl driven by MSCVneo EB (24). Results, plotted as a fraction of the OD570–630 obtained in the same clones using 5% IL-3-containing WEHI conditioned medium, denote the mean of three experiments. Error bars denote standard deviations.

FK1012 stimulates expansion of genetically modified bone marrow cells. Results from two independent experiments (A and B) are shown. After retroviral transduction, marrow cells were cultured in IMDM containing 10% FCS, in the presence or absence of FK1012 (100 nM) (A and B) or flt-3 ligand (100 ng/ml) (A). Cells were counted at various times during culture. (⋄) No FK1012, no FL; (♦) FK1012 alone; (○) FL alone; (•) FK1012 plus FL. Note that the y axis is on a logarithmic scale.

FK1012-responsive cell lineages. (A) Wright–Giemsa stain after 9 days of culture in FK1012. Neutrophils (n) and macrophages (m) are abundant. Small numbers of megakaryocytes (M) also can be seen. (B) Wright–Giemsa stain after 42 days of culture in FK1012. Neutrophils have disappeared, and megakaryocytes emerge as the predominant phenotypically identifiable cell population. (C) Anti-CD41 alkaline phosphatase staining of cells cultured for 42 days in FK1012. Megakaryocyte cytoplasm stains red and nuclei stain blue. Note that the full spectrum of megakaryocyte maturation is observed, from small, blast-like cells to large, polyploid cells.

CFU-C assays at various time points during suspension culture. Cells were harvested on the days indicated and cultured in semisolid media either in the presence or absence of G418. The concentration of G418, 800 μg/ml, was sufficient to prevent the growth of nontransduced cells (data not shown). Solid bars, CFU-C numbers in the presence of G418; open bars, CFU-C numbers in the absence of G418. (A) No FK1012, no FL. (B) FK1012 alone. (C) FL alone. (D) FK1012 plus FL. Note that FK1012-mediated CFU-C expansion markedly favors the genetically modified cell population.