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. 2007 Jun 18;35(13):4359–4368. doi: 10.1093/nar/gkm444

Figure 3.

Figure 3.

(A) Upper panel shows a schematic representation of the pES hybrid minigene carrying the CFTR exon 9 along with intronic flanking sequence. Black, shaded and white boxes represent alpha globin, fibronectin and CFTR exons, respectively. The arrows indicate the position of the primers used to amplify the processed mRNA species. The dotted lines represent the expected splicing pattern that determines exon 9 inclusion/exclusion. Lower panel shows a close-up view of the nucleotide composition of the IVS9 sequence in the PstI/KpnI/NdeI cloning region. The black box represents the exact position in which the AS1 + AS2 (pTB AS1 + AS2PK) and AS3 + AS4 (pTB AS3 + AS4PK) sequences were inserted. (B) Shows a comparison of the RT–PCR splicing profiles of these two constructs together with the pES and TG11T5 reference minigenes following transfection in Hep3B cells. The position of the transcripts including exon 9 (ex9+) and lacking exon 9 (ex9−) are marked on the right. The results of three independent experiments were quantified using radioactive PCR and are reported in (C) with SD values. (D) Shows a set of analogous transfections in Hep3B cells using a set of plasmids in which each individual AS1, AS2, AS3 and AS4 sequences was inserted in the PstI/KpnI sites of the pES plasmid (pTB-AS1PK to pTB-AS4PK). A quantification of the effect of these sequences on CFTR exon 9 inclusion is reported in (E).