Rescue of FVII splicing by modified U1-snRNAs in Hep3B cells. (A,B) Electrophoretic separation on 3% agarose gel, and direct sequencing, of RT-PCR products obtained from total RNA of cells transfected with the expression vectors for normal (Wt) or mutated (9726 + 5a) minigenes, and equimolar concentrations of plasmids encoding for the modified U1-snRNAs. The separation on 12% polyacrylamide gel of the amplified transcripts on U1 + 5a expression and the sequence of the correctly spliced form are also shown in the lower part of panel B. The scheme of transcripts (1, 2, 3) and the primers used (arrows) are depicted on the right side. The additional band observed for the 9726 + 5a mutant (A) corresponded to heteroduplex (H.D), as demonstrated by sequencing. Fragment sizes in panel A were: 637 bp (1), 600 bp (2), and 480 bp (3). Fragment sizes in panel B were: 228 bp (1) and 191 bp (2). M indicates molecular weight marker. (C) Separation on a denaturing capillary system (automated ABI-3100) of fluorescently labeled RT-PCR products obtained from total RNA of cells expressing the Wt (i) or the 9726 + 5a minigene without (ii,iii) and on (iv,v) overexpression of the U1 + 5a snRNA. The scheme of transcripts, and of primers used (arrows), is depicted on the top. Separation of 1 μL of 1:100 diluted RT-PCR reaction is shown, with the exception of panel Ciii) in which 1 μL of the 1:20 dilution was loaded (overloaded). The peaks, which might suggest the presence of a doublet, were referred to as single bands by the automated ABI-3100 system. As expected, the fragment sizes of the normal and aberrant transcripts were 236 bp and 273 bp, respectively.