Splicing functional assays of a single minigene with eight exons of the BRCA2 gene

Abstract

Splicing disruptions is one key pathogenic mechanism in inherited diseases. We are currently investigating the contribution of aberrant splicing of BRCA1/2 genes to hereditary breast/ovarian cancer. A powerful approach to study the splicing outcomes of DNA variants is a splicing reporter minigene especially when patient RNA is not available. We constructed a single minigene of 8 BRCA2 exons (19 to 26) in a pSPL3-derived plasmid in 5 cloning steps, which is, as far as we know, the largest BRCA2 minigene ever reported. The genomic fragment from exons 19 to 26 is more than 27 kb in length that was reduced to a final insert of 4.7 kb with internal deletions of introns 20, 21, 24 and 25.This construction was transfected in HeLa cells and we observed a main RNA isoform of the expected size of 1.5 Kb that contained the vector constitutive exons and BRCA2 exons 19 to 26. Several splicing variants of each exon were generated in the wild type minigene by PCR mutagenesis and assayed to demonstrate the usefulness and reliability of this large construction. Splicing reporter minigenes are straightforward and robust tools to distinguish between pathogenic mutations and innocuous variants. The use of minigenes with several exons facilitates the analysis of putative splicing variants in a single minigene and emulates the physiological genomic context where the splicing reactions take place.