Rescue of FVII splicing and FVII function by the modified U1+5a in COS-1 cells. (A) FVII-mediated FXa generation (grey histograms) and FVII coagulant (white histograms) activity in conditioned medium from COS-1 cells transfected with pSCFVII-wt or the pSCFVII-9726+5a without and with equimolar concentration (1×) or an excess (1.5×) of pU1+5a. Mean and SD are shown. The PT-based assays have been performed on an ACL TOP automated coagulometer (Instrumentation Laboratory, Milan, Italy). Briefly, 50 μL conditioned medium was mixed with 50 μL FVII-depleted plasma (Instrumentation Laboratory) and incubated for 30 seconds at 37°C. RecombiPlasTin 2G (100 μL; Instrumentation Laboratory), as source of recombinant human tissue factor, calcium, and phospholipids were then added and coagulation time recorded. nd indicates not detectable. (B) Separation on a denaturing capillary system (automated ABI-3100) of fluorescently labeled RT-PCR products obtained from total RNA of cells transfected as indicated in panel A. A representative example is shown. The scheme of transcripts, and of primers used (7F^F, fluorescently labeled; 8R),¹²  is depicted at the bottom. Separation of 1 μL of 1:100 diluted RT-PCR reaction is shown. As expected, the fragment sizes of the normal and aberrant transcripts were 236 bp and 273 bp, respectively.