Design of a GFP reporter for splicing analysis in mammalian cells

Abstract Objective: The great majority of eukaryotic genes are formed by exons and introns. Pre-RNA transcripts are extensively processed in the nucleus, with the addition of a cap group at the 5′ end, intron removal and exon ligation (splicing) followed by addition of a poly-A tail at 3′ end. Splicing is performed by specialized macromolecular machinery named spliceosome, composed of five small ribonucleoprotein particles (snRNPs) and several proteins. The activity of this complex is highly accurate due to coordinated activity of its components. Altered splicing has already been related to the development of several diseases as amyotrophic lateral sclerosis and different types of cancer. Detailed understanding of splicing regulation in eukaryotic cells can be achieved using splicing reporter systems. Results: We designed a new splicing reporter plasmid suitable for analysis in mammalian cells. Our reporter is based on splicing of the GFP sequence. The greatest advantages of this system are the ease of visualization of the splicing outcome, by using a fluorescence microscope to confirm GFP expression from the reporter. Also, quantification of splicing efficiency using real-time PCR is possible. The use of this system allows rapid and easy detection of the splicing results in cultured cells.