Figure 1.

Splicing assay with the exon trapping assay. (a) Electrophoresis of cDNA-PCR products generated from the wild-type and mutant constructs (exon 20 from patient 1 and exon 5 from patient 2) after transfection into COS-7 cells. Lane 1: DNA marker, GeneRuler DNA Ladder Mix (MBI Fermentas, Glen Burnie, MD, USA); lane 2: splicing product of exon 20 wild-type construct; lane 3: splicing product of exon 20 mutant construct; lane 4: splicing product of exon 5 wild-type construct; lane 5: splicing product of exon 5 mutant construct; lane 6: splicing product of pSPL3. In the absence of an inserted fragment, a vector/vector splice product of 263 bp is produced (A: lane 6, B: β splice product). In the presence of an inserted wild-type fragment, splicing of the vector gives rise to two products: a vector/vector product of 263 bp and a correctly spliced vector/genomic product containing the inserted exon (237 or 132 bp; A: lanes 2 and 4, B: α). The abundance of vector/genome product as compared with the vector/vector product indicates that no enhancement of exon 20- or 5-skipping occurred in the wild-type constructs. Mutant constructs did not produce any detectable vector/genomic product from the mutant inserts obtained from patients 1 and 2 (lanes 3 and 5), indicating complete splicing-out of exons 20 or 5. (b) Genomic DNA constructs showing the cloned sequence within the exon-trapping vector, pSPL3. The sizes of vector DNA (white box), intronic DNA (lines) and exonic DNA (black box with white dots) are indicated. Two possible splicing pathways, α (vector/genome) and β (vector/vector) are shown with their respective spliced products.