Ex vivo splicing correction in patient’s megakaryocytes. Hematopoietic progenitor cells were isolated from peripheral blood and differentiated toward the megakaryocytic lineage as described in “Methods.” At day 9 of culture, the FV-deficient patient’s cells were either left untreated (A) or treated with rescuer MO or with the U7-SmOPT construct expressing rescuer U7snRNA at the indicated concentrations (B-E). Untreated cells from a normal control are shown for comparison (F). After immunofluorescence staining with Hoechst 33258 (cell nuclei, in blue) and with a FITC-labeled anti-mouse antibody recognizing the primary anti-FV antibody (FV, in green), cells were examined under a Leica DMI6000CS fluorescence microscope using a 63×/1.40 oil-immersion objective at room temperature. Images were acquired using a DFC365FX camera and analyzed with Leica LAS-AF 3.1.0 software. Each panel represents the overlay of 2 images of the same preparation stained with Hoechst 33258 and FITC-labeled antibody, respectively. Four representative microscope fields are shown for each condition. Following isolation and reverse transcription of total RNA, the F5 mRNA splicing pattern in untreated and treated patient’s megakaryocytes was analyzed by real-time qPCR (G). Results are expressed as the ratio between the aberrant and normal F5 mRNA, plotted on a logarithmic scale. Untr., untreated; R-MO, rescuer MO; R-U7, rescuer U7snRNA.