F36VFGFR1 supports the expansion of transduced mouse bone marrow cells in culture. (A) Schematic depiction of F36VMpl and F36VFGFR1 retrovirus vectors. LTR indicates MSCV long terminal repeat; GFP, green fluorescent protein; IRES, encephalomyocarditis virus internal ribosomal entry site; ▪, myristylation domain; ▧, F36V-modified FKBP12; and red and maroon boxes, cytoplasmic domains of mouse Mpl and rat FGFR1. At the carboxyterminal end of both F36VMpl and F36VFGFR1isan epitope tag from the HA protein of influenza³¹  (not shown). (B) Colony assays of marrow cells transduced with the F36VMpl or F36VFGFR1 vectors performed in the presence (GF) or absence (–) of a combination of growth factors, or in the presence of 100 nM AP20187 without growth factors (AP). Total colonies (bars) and GFP⁺ colonies (▦) are indicated. (C) Logarithmic expansion of F36VFGFR1-transduced marrow cells over time in serum-containing cultures plus AP20187 (100 nM) without added cytokines. Cell growth ceased following CID withdrawal (–AP, open symbols). We have previously reported a similar exponential expansion of mouse marrow cells in response to CID-activated derivatives of Mpl,^10-14  but not CID-activated derivatives of Flt3 or G-CSF receptor.¹²  (D) Rise in the percentage of GFP⁺-expressing F36VFGFR1-transduced marrow cells over time in culture with CID. (E) Differences in cell types emerging from cultures of F36VFGFR1-transduced (•) versus F36VMpl-transduced (○) marrow cells. Similar results were obtained in 7 independent experiments using F36VFGFR1, and in previously published reports using F36VMpl.¹⁰  (F) Phase-contrast microscopy (left panels) and Wright-Giemsa staining (right panels) (× 200) of F36VMpl (top) or F36VFGFR1 (bottom) transduced lin^–c-kit⁺, sca-1⁺ cells after 6 days (phase contrast) or 8 days (Wright-Giemsa) of culture in AP20187. Phase-contrast images were visualized using a Leica DMIL inverted microscope (Leica, Wetzlar, Germany) equipped with a C PLAN L 20×/0.30 numerical aperture (NA) PH1 objective. Wright-Giemsa stains were visualized using a Leica DMLB microscope equipped with an HC PL FLUOTAR 20×/0.50 NA objective. Images were captured using a Leica DFC480 digital camera and Leica Image Management software, and were processed using Adobe Photoshop software version 8.0 (Adobe Systems, San Jose, CA). (G) Flow histograms of lin^–c-kit⁺, sca-1⁺ cells from mice that received transplants 4 months earlier with F36VMpl-(top) or F36VFGFR1 (bottom)–transduced marrow cells. Expression levels of GFP and the HA-tagged fusions overlap significantly, but average 1.6-fold higher for F36VFGFR1. Values indicate mean fluorescent intensity (MFI). Red lines in the left panels indicate GFP^– cells. Blue lines in the left panels, GFP⁺ cells; red lines in the right panels, cells stained with isotype control antibody; and the blue lines in the right panels, cells stained with HA antibody.