(C) Confocal microscopy of the transfectedĬells. The p value wasĭetermined by the two-tailed Student's t-test Western blot measured by ImageJ software. The amount of each form was estimated from its band intensity on the CleavageĮfficiency = cleaved form/(cleaved form+uncleavedįorm).
(B) Quantitation ofĬleavage efficiency of indicated 2As. Asterisks indicate two major byproducts.Īnti-β actin antibody was used as a loading control. The cleavage efficiency wasĪssessed using GFP and DsRed antibodies to decorate NLS-EGFP and Were processed for WB 24 hr post-transfection. Of cleavage efficiency of the 2As in HEK293T cells. HEK293T cells were transfected with the indicated plasmids. 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. 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. 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. 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. 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. 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. Among various strategies employed to construct bicistronic or multicistronic vectors, an internal ribosomal entry site (IRES) has been widely used. When expression of more than one gene is required in cells, bicistronic or multicistronic expression vectors have been used.
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