FIGURE 3.

Minigene constructs for splicing pattern investigation. (A) Schematic representation of the GLA minigene construct, extending from exon 5 to exon 6. The nucleotide sequences of the wild type (WT) and mutant (MT) minigenes are shown below. The changed nucleotide in intro 5 of both wild type and mutant GLA minigene are labeled by underscore. (B) Minigene splicing assay. GLA minigenes harboring the wild type or mutant GLA pseudoexon were transiently transfected into HEK293T cells. After RNA isolation the splicing products were analyzed by RT-PCR. The lower bands represent correctly spliced exons, whereas the higher band represent the GLA pseudoexon inserted 36 nt from intro 5 between minigene exons 5 and 6. (C) Western blot analysis for the expression of the wild type and mutant pEGFP-GLA-minigenes. Whole cell lysates were separated by SDS–PAGE (12% acrylamide). GFP monoclonal antibodies were used.