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.
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