Fast Cloning of Splicing Reporter Minigenes

The most common technique used to analyze the regulation of an alternative exon is through reporter minigene constructs that are transfected into cells. Their construction by conventional cloning methods can be time-consuming, however, and is often complicated by the limited availability of appropriate restriction sites. An alternative fast and simple recombination-based method is described for the generation of splicing reporter genes, using homologous recombination. The appropriate recombination sites are introduced into the gene fragment to be analyzed by PCR; this fragment is then recombined with the pSpliceExpress series of vectors. The resulting construct is an exon trap vector that contains the fragment of interest flanked by two constitutively spliced exons. The system allows the generation of minigenes within one week.