Minigenes to Confirm Exon Skipping Mutations

Although several bioinformatic tools exist to predict the effect on splicing of a nucleotide change, experimental verification with minigenes is essential for diagnostic purposes, as well as for revealing disease mechanisms and monitoring therapeutic interventions. Minigenes are splice reporter vectors (also known as exon-trapping vectors) that allow confirmation of the effect of mutations on the splicing process, indicated when patients’ samples for RNA studies are not available. The minigene vector codes for exonic portions of a gene defined by functional 5′ splice donor and 3′ splice acceptor sites separated by intronic sequences where a polylinker is located. Here, the exon carrying the mutation under study is cloned along with its flanking intronic sequence. The resulting construct, in its wild-type and mutant sequence version, is transfected in established cell lines and the vector splicing pattern is analyzed. Ideally, the wild-type minigene results in correct exon inclusion, while the mutant construct results in exon skipping or other aberrant transcripts.