Abstract
When expression of more than one gene is required in cells, bicistronic or multicistronic expression vectors have been used. Among various strategies employed to construct bicistronic or multicistronic vectors, an internal ribosomal entry site (IRES) has been widely used. Due to the large size and difference in expression levels between genes before and after IRES, however, a new strategy was required to replace IRES. A self-cleaving 2A peptide could be a good candidate to replace IRES because of its small size and high cleavage efficiency between genes upstream and downstream of the 2A peptide. Despite the advantages of the 2A peptides, its use is not widespread because (i) there are no publicly available cloning vectors harboring a 2A peptide gene and (ii) comprehensive comparison of cleavage efficiency among various 2A peptides reported to date has not been performed in different contexts. Here, we generated four expression plasmids each harboring different 2A peptides derived from the foot-and-mouth disease virus, equine rhinitis A virus, Thosea asigna virus and porcine teschovirus-1, respectively, and evaluated their cleavage efficiency in three commonly used human cell lines, zebrafish embryos and adult mice. Western blotting and confocal microscopic analyses revealed that among the four 2As, the one derived from porcine teschovirus-1 (P2A) has the highest cleavage efficiency in all the contexts examined. We anticipate that the 2A-harboring cloning vectors we generated and the highest efficiency of the P2A peptide we demonstrated would help biomedical researchers easily adopt the 2A technology when bicistronic or multicistronic expression is required.
Highlights
In biomedical research, the simultaneous expression of more than one gene in cells or organisms using a single plasmid is sometimes required
We show that among four different 2A peptides, a 2A peptide derived from porcine teschovirus-1 (P2A) has the highest cleavage efficiency in three human cell lines, zebrafish embryos and mouse liver
We did not test if this is the case, it is very likely that the cloning vectors would act in a similar manner in Xenopus, because an simian cytomegalovirus (sCMV) promoter functions in Xenopus as well [15] and the 2A-mediated cleavage occurs in all eukaryotic cells [9]
Summary
The simultaneous expression of more than one gene in cells or organisms using a single plasmid is sometimes required To this end, several strategies have been employed: (i) multiple promoters fused to the genes’ open reading frames (ORFs); (ii) insertion of splicing signals between genes; fusion of genes whose expressions are driven by a single promoter; (iii) insertion of proteolytic cleavage sites between genes; and (iv) insertion of internal ribosomal entry sites (IRESs) between genes [1,2].
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