Figure 3. Evaluation of the effect of PRPF31 c.1146+5G>T on splicing in vitro. A, B: Minigene constructs and products obtained in the in vitro splicing assay. Shown is a schematic representation of the constructs (A), which include PRFF31 exons 10, 11, and 12 (represented by boxes) and the introns between them. Either G (wt) or T (mutant) is present at position +5 of intron 11. The locations of the primers used for the RT-PCR analysis are also shown (indicated by arrows). Constructs were transfected into COS-7 cells, followed by RNA extraction and RT-PCR analysis (B). cDNA derived from the wild-type (wt) construct yielded two splice products: one harboring exons 10, 11, and 12 and the other showing the skipping of exon 11. cDNA derived from the mutant construct mainly yielded the splice product lacking exon 11. mut, mutant; M, size marker.