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. 2016 Jun 6;126(7):2575–2587. doi: 10.1172/JCI85830

Figure 4. Splice donor site mutations lead to cryptic splice site use or exon skipping.

Figure 4

(A and B) Splicing construct minigenes were generated by incorporating genomic regions of the TEK gene into the pSPL3 vector via XhoI and BamHI restriction sites. Vector exons, V1 and V2, are depicted as black boxes and TEK exons 5, 6, and 22 are in gray. WT and mutant splicing products, with included exon sizes in base pairs, are indicated by dashed lines above and below the construct, respectively. The locations of the splice site mutations are shown (*). (A) WT (WT-5) and mutant (M-5) genomic fragments containing TEK exons 5 and 6 were used to model the c.760+2T>C mutation from family 7. (B) WT (WT-22) and mutant (M-22) genomic fragments containing TEK exon 22 were used to model the c.760+2T>C mutation from family 8. (C) Gel electrophoresis of RT-PCR products from transfected Cos-7 cells. Vector exon-specific primers are indicated by arrows in A and B. TF -ve, cells transfected with PBS only; PCR -ve, PCR-negative control. WT and mutant transcript content, determined by Sanger sequencing, is depicted to the right of the gel image. The additional 21 bp of intron 5 sequence identified within the M5 transcript is shown incorporating a premature termination codon between exons 5 and 6.