Fig 5.

The GPT2 synonymous mutation c.1035C>T (p.G345=) results in aberrant use of a donor splice site. a Generation of an exon trapping vector for testing of the effects of the c.1035C>T (p.G345=) mutation on splicing. Exon 8 of the human wild-type GPT2 gene, as well as portions of the flanking introns (141 bp of intron 7 and 290 bp of intron 8), was ligated into the multiple cloning site (MCS) of the pET01 vector (mutated base in red). Site-directed mutagenesis was performed to generate a pET01-GPT2 vector with the c.1035C>T mutation in exon 8. Wild-type (WT) and mutant (Mut) vectors were confirmed by Sanger DNA sequencing (bottom chromatograms). The mutated base is underlined. b,c Results of exon trapping assays. RT-PCR were performed with cDNA generated from HEK293T cells transfected with either the wild-type (WT), mutant (Mut), or control (empty pET01) vector. Amplicons were separated by agarose gel electrophoresis (b) and analyzed by Sanger DNA sequencing (c). The c.1035C>T mutation in exon 8 results in a 4-bp deletion due to creation of a new splice donor site (compare bottom two chromatograms)