Co-expression of multiple genes in a desired ratio is attractive for a wide range of basic and biomedical applications including cellular reprogramming1,2,3,4,5, expression of multimeric proteins in gene therapy6,7,8, expression of fluorescent proteins for live cell imaging9,10,11 and genome editing12,13. Several strategies have been developed for multigene co-expression, such as the use of internal ribosome entry sites (IRES) and polycistronic vectors, proteolytic cleavage between genes, and “self-cleaving” 2A sequences14,15.
The t2a peptide is a highly conserved 18-22 amino-acid viral oligopeptide that mediates ribosome skipping and translational silencing by binding to the ribosomal A site and forming a steric hindrance with the adjacent protein sequences. A 2A-cleaved fragment and a complete, unglycosylated polypeptide are subsequently released from the ribosome. The cleaved fragment is subsequently degraded by the host cell proteases. The process is reversible, and the protein synthesis can resume once the 2A-cleaved fragment is removed from the ribosome.
In this study, we created a bi-cistronic vector in which a 66 bp P2A-ssRFP fragment was fused with the sporozoite surface antigen 13 (SAG13) promoter and 3′ UTR followed by RFP and EYFP. A 17 aa Ebola epitope peptide derived from the Zaire strain glycoprotein (GP; ATQVEQHHRRTDNDSTA) was included in the fusion for specific cleavage by a human endoplasmic reticulum (ER) lectin-like protein. The resulting plasmid was transfected into Ebola virus Mayinga-strain sporozoites and analyzed by flow cytometry for the presence of GFP-positive and EYFP-positive cells.
The results show that positional effects of 2A on gene expression are more prominent than differences in the 2A sequence itself. We observed that GFP fluorescence intensity at position 2 in tri-cistronic constructs containing different combinations of P2A and T2A was not affected by the order of the two genes, but that positional effects were exacerbated in quad-cistronic constructs with P2A inserted at positions 1, 2, and 4.