Interruption of coding sequences by heterologous introns can enhance the functional expression of recombinant genes

Sustained expression of recombinant proteins is a critical factor for the e ffectiveness of numerous applications in the biomedical sciences including the treatment of human disease by gene therapy, the large scale production of therapeutic proteins, as well as the investigation of gene function by t ransgenesis or cell type specific mutagenesis. Although much attention has been paid to the optimisation of regulatory sequences such as promoters, un translated regions and polyadenylation signals, effective and sustained exp ression of recombinant genes in vivo is often difficult to achieve. Here we report that the creation of artificial exons, by insertion of two short he terologous introns into open reading frames, is not only compatible with fu nctional expression, but also leads to a 30-fold enhancement of mRNA produc tion for both green fluorescent protein and the bacteriophage P1-derived Cr e recombinase. The levels of green fluorescence were increased five-fold in cell lines and sustained long-term expression at increased levels was obse rved in rat brain after transduction with a herpes simplex virus-based vect or The data presented identify a means by which the expression of recombina nt genes can be enhanced considerably, in addition to and independently fro m the surrounding regulatory sequences. The method should help obtain susta ined and effective expression of recombinant proteins in vivo.