BclxL transgene expression in megakaryocytes.
(A) PCR-based cDNA amplification, showing transgene expression in BM of transgenic mice, as described in “Materials and methods.” Reverse-transcribed, amplified DNA was electrophoresed, transferred onto a nylon membrane, and analyzed with 32P-labeled probes as indicated. The reaction was carried out in the absence or presence of reverse transcriptase (RT) for transgenic lines (Tg) and wild-type (WT) mice. SV40 probe detects the transgene only, BclxL probe detects the endogenous and transgenic BclxL at similar positions, and both PF4 and GAPDH are used to evaluate and normalize for the efficiency of the RT reaction. The lower panel with SV40 probe shows twice the time of the exposure of the film as the top panel to permit visualization of the band in Tg12. Data are representative of 3 experiments. (B) BM cells derived from transgenic (Tg) or control (WT) mice were cytospun (as a dense layer of cells to allow focus on the rare megakaryocytes) and Cy3-labeled with rabbit antibodies to human BclxL (which also reacts with mouse BclxL). Transgenic megakaryocytes, identified based on size and morphology, displayed higher levels of BclxL, as compared with control (examinations were done blindly). The staining appeared cytosolic, as also described by others.42 The data are representative of 2 transgenic lines (2 mice per line in duplicates; original magnification, × 400). (C) Freshly derived BM cells were cytospun onto a slide and subjected to immunohistochemistry with anti-BclxL (middle panel). Megakaryocytes were identified based on staining with an antibody to CD41 (top panel) and DNA was stained with Hoechst (lower panel). No staining was observed with secondary antibody alone and no BclxL-specific fluorescence was observed on staining with CD41 alone and vice versa (not shown). The micrographs are of a representative field, further demonstrating enhanced, lineage-specific expression of BclxL in a transgenic line (original magnification, × 400).
