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. 2021 Feb 28;12(3):353. doi: 10.3390/genes12030353

Figure 3.

Figure 3

Splicing minigene reporter assay based on the pSPL3 exon-trapping vector. (A) Schematic representation of the pSPL3 minigene reporter used for the molecular characterization of APC splicing mutation c.1621_1626+7del. The pSPL3 vector contains two exons (SD and SA) and a functional intron, with transcription beginning after the SV40 promoter and ending at the late poly(A) signal (LPAS). EcoRI and BamHI indicate the cloning sites used to subclone the genomic APC fragments obtained from the wild type and mutant alleles (c.1621_1626+7del). (B) RT-PCR analysis of transcripts derived from the indicated pSPL3 reporter minigenes transfected in HEK-293 cells. Left: Agarose gel electrophoresis showing the RT-PCR products obtained with the SD6 and SA2 primers from HEK-293 cells transfected with the pSPL3 empty vector (263 bp), the pSPL3 vector with the genomic APC fragment from the wild type allele (341 bp), or the pSPL3 vector with the genomic APC fragment from the mutant allele (263 bp), and untransfected HEK-293 cells (negative control). Center: Schematic diagrams showing the RT-PCR products obtained. Right: Sequencing electropherograms of the RT-PCR products obtained.