Fig. 5

Splicing analysis using Exontrap pET01. a Diagram of LRAT genomic DNA, the location of the c.541-15T>G mutation and the fragment inserted into pET01 construct. In the pET01 construct, the multiple cloning site (MCS) is in an intron flanked by pre-proinsulin 5′ and 3′ exons. The primers used for cDNA amplification are within the pre-proinsulin 5′ and 3′ exons and indicated by black arrows. b Diagrams of transcript splicing of WT- and Mut- LRAT-pET01. Splicing events in the WT construct are shown by a blue line, and transcript splicing of c.541-15T>G Mut-LRAT-pET01 construct are shown by a red line. Results derived from in silico prediction (top), results obtained from minigene assay using pET01 vector (middle), and results obtained from minigene assay using pET01 vector with the splice acceptor site of 3′ exon abolished (bottom). c Electrophoresis results of cDNA amplification obtained from 293T cells transfected with empty construct pET01, wildtype construct (WT), c.541-15T>G mutant construct (Mut), modified empty construct (pET01-2), modified wildtype construct (WT-2), and modified c.541-15T>G mutant construct (Mut-2). The major PCR products of cDNA amplification from each construct as verified by sequencing are indicated in d