F5 minigene construct. (Top) Schematic representation of the F5 minigene construct. The pTB vector contains the promoter (black arrow) and exons 1 to 3 of the human α-globin gene (gray boxes) as well as exons 24 to 25 of the fibronectin-1 gene (black boxes). High transcription levels are ensured by an SV40 enhancer (not shown). The F5 minigene, encompassing the 3′ end of intron 7, exon 8, intron 8 (with or without the +268A>G mutation), exon 9 and the 5′ end of intron 9 of the F5 gene, was inserted between the vector’s unique Sac II and Nde I restriction sites. The position of the c.1296+268A>G mutation is marked by a star. The dotted box indicates the intronic pseudo-exon (ψ). (Bottom) Mature transcripts produced by the F5 minigene construct. The black arrows represent the primers used for qualitative analysis of the F5 mRNA; the white arrows represent the specific forward primers used to distinguish between the 2 differently spliced transcripts using real-time qPCR. EX, exon; IVS, intervening sequence (intron); Prom, promoter.