Generation of PF4-tTA-VP16 transgenic lines and confirmation of transgene expression. (A) Transgene integration. PCR of genomic DNA was used to screen for specific transgene integration (yielding a 460-bp product shown here) following protocols detailed in “Materials and methods.” Three founder lines, PF4-tTA-VP16 (referred to as PF4-tTA) nos. 38, 42, and 54, were identified, as confirmed by Southern blot analysis. (B-C) Transgene expression. After obtaining homozygous PF4-tTA-VP16 lines, we used Western blot analysis (B) to identify a line with the highest expression of the tetracycline responsive enhancer fusion protein (tTA-VP16). Bone marrow cells were subjected to Western blotting with anti-VP16 antibody to detect expression of tTA-VP16. Actin levels were determined to confirm equal loading of samples. Tissue-specific expression of tTA-VP16 was determined by immunohistochemical analysis of bone marrow cells (C) using anti-VP16 as a primary antibody and FITC-conjugated anti–mouse IgG as a secondary antibody. Since transgenic line no. 42 displays high expression of tTA-VP16 fusion protein in megakaryocytes (indicated by arrows), we decided to use this line for subsequent crossbreeding and experiments. Shown is a representative experiment in which the cells were viewed at a magnification of × 20. Images were captured using an Olympus ×70 microscope with Hamamatsu CCD camera C4742-95. Images were acquired with Openlab 3.1.2 software.