Skipping of exon 11 requires sub-optimal splice sites. Exons 10, 11, and 12 and the sequences of the splice sites at the ends of the introns are shown. Panel A: The 5' splice sites of exon 10 and exon 11 are 5/7 and 4/7 matches to the consensus sequence, respectively. The splice site scores (out of 100) are indicated above each site. These sites were altered to match the consensus sequence in the parental IR minigene (pIRB) that contains 2.3 Kb of intron 10 and 360 bp of intron 11 giving minigenes pIRB.MC15 and pIRB.MC1. The 3' splice sites of exon 11 and exon 12 contain a number of purine residues interspersed in the polypyrimidine tracts. These residues were mutated to thymidines to strengthen each 3' splice site giving minigenes pIRB.MC0 and pIRB.MC11. Mutated residues are shown in bold and underlined. The intervening sequence is omitted and indicated by the slashes. The minigenes were transfected into HepG2 and HeLa cells. RNA was extracted and mRNA subjected to reverse transcription and amplification by PCR. The columns to the right show the percentage of skipping of exon 11 in either cell line. Results are the mean of four independent experiments. Asterisks indicate statistical significance versus the parental minigene pIRB (*p < 0.05, ***p < 0.001). Panel B: The same mutations were introduced into the splice sites of intron 10 in minigenes containing internal deletions of intron 10. Minigene pIRA contains the purine-rich enhancer (GA) and the inhibitor (INH), minigene pIRK only contains the inhibitor (INH). Sequences of the 5' and 3' splice sites of intron 10 are shown. Intervening sequence is indicated by dashed lines. Both pIRA and pIRK contain the identical intron 11 and exon 12 sequences as pIRB in Panel A but these sequences are omitted for clarity. These minigenes were transfected into HepG2 and HeLa cells as above. The percentage of skipping of exon 11 is given in the columns on the right. Results are the mean of four independent experiments. Asterisks indicate statistical significance versus the minigene with natural splice sites * p < 0.05).