Conditional and reversible expression of transgenes in megakaryocytes. (A) Induced β-galactosidase expression. Shown are immunohistochemistry analyses of freshly isolated bone marrow cells from double-transgenic littermates PF4-tTA-VP16/CMV–Aurora-B/β-gal no. 41/42 shown in Figure 3B and age- and sex-matched wild-type mice. Tet-off mice were removed from doxycycline treatment (referred as Tet-off to keep with a terminology that refers to tetracycline inducible promoter) 4 weeks prior to analysis, whereas Tet-on mice were continuously supplemented with doxycycline. To detect β-galactosidase (β-gal)–induced expression, mouse monoclonal antibody against E coli β-gal was used (red). The same preparations were also stained for CD41 (specific marker for megakaryocytes) using anti–CD41-FITC (green). Images were captured with an Olympus microscope using a × 60 objective. DAPI indicates 4,6 diamidino-2-phenylindole. (B) Reversible transgene expression. To demonstrate that conditional gene expression was reversible, half of the Tet-off mouse population received doxycycline for 4 weeks (Bvi), while the other half of its littermates remained Tet-off (Bii). Similarly, half of the Tet-on transgenic mouse population (doxycycline supplement) was removed from doxycycline treatment for 4 weeks (Bv), while the other half remained on doxycycline supplement (Bvi). β-galactosidase was followed as in panel A (not shown) as well as by enzyme activity (shown here as a representative). Fresh bone marrow cells derived from the double-transgenic mice (PF4-tTA-VP16/CMV–Aurora-B/β-gal no. 41/42) and equivalent wild-type cells were stained for β-gal as outlined in “Materials and methods” (visualized via phase-contrast, × 40 original magnification). Megakaryocytes were clearly identified based on their size and morphology. Images were captured using an Olympus ×70 microscope with Hamamatsu CCD camera C4742-95. Images were acquired with Openlab 3.1.2 software.