Phenotype of primary bone marrow cells from

GPIIb-tva⁺mpl^−/− mice after infection with RCAS-Mpl and selection in TPO. (A) TVA staining of bone marrow from GPIIb-tva⁺mpl^+/+mice and GPIIb-tva⁺mpl^−/−mice cultured as described in “Materials and methods” demonstrates the anticipated reduction in TVA-expressing cells in thempl^−/−background. Arrowheads indicate mature megakaryocytes, and arrows indicate TVA-expressing, small, early megakaryocyte-lineage cells. (B) Wright-Giemsa stain demonstrates the irregular cell shape and the folded nuclei (left panel), and immunohistochemical staining demonstrates surface CD41 (center panel) and intracellular vWF (right panel) inGPIIb-tva⁺mpl^−/−bone marrow cells after infection with RCAS-Mpl and selection in TPO. (C) FACS analysis for surface antigen expression on the cells in panel B demonstrates expression of the megakaryocyte marker CD41 but no expression of erythroid (Ter119), T-lymphocyte (CD3), B-lymphocyte (B220), granulocyte (Gr-1), or macrophage (F4/80) markers. The FLAG-tagged Mpl introduced by RCAS-Mpl is readily detected on the cell surface with an anti-FLAG antibody. Thin lines, fluorochrome-conjugated isotype controls; thick lines, fluorochrome-conjugated antigen-specific antibody.