Intron 2 of human beta-globin in 3′-untranslated region enhances expression of chimeric genes

The possibility of enhancing heterologous gene expression in mammalian cells by the introduction of an intron in 3′ untranslated region (UTR) was investigated. To this end, a fragment of human betaglobin gene with intron 2 and flanked exon regions was introduced into the vector-encoding green fluorescent protein TagGFP2 after the TagGFP2 stop-codon (Int+). The distance between the stop-codon and the exon junction was 35 nucleotides. It ensured that Int+ mRNA was resistant to degradation by nonsense mediated decay (NMD) machinery. A control vector Intcontained corresponding intronless sequence of the beta-globin mRNA. On the same plasmid, the second gene encoded far-red fluorescent protein Katushka was used to normalize fluorescence for transfection efficiency and expression level in individual cells. Transiently transfected HEK293T cells were analysed by flow cytometry. It was shown that cells transfected with plasmid carrying the Int+ gene possess 1.8 ± 0.2 fold higher green fluorescence compared to Intcells. The observed effect was used to enhance expression of destabilized variants of yellow fluorescent protein TurboYFP-dest with high degradation rate in mammalian cells. We believe that introduction of beta-globin intron in the 3′-UTR of the chimeric gene can be used to enhance its expression and may be advantageous in some cases when usage of 5′ UTR intron is inappropriate.