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.

The prevention of undesired abdominal wall adhesion after surgery was investigated using asymmetric polysaccharide composite membranes as physical barriers. The hyaluronate (HA) and chitosan (CS) based asymmetric composite membranes were prepared with one smooth surface to inhibit fibroblasts and one porous surface to improve the tissue cells. With adaption of the reaction temperature, pH and component ratios, a series crosslinked membranes were processed with different appearance, mechanical strength and swelling ratios. Cell culture results showed the fibroblasts were inhibited on the composite membranes with comparison of that on petri dish. The osteoblasts proliferated obviously on the porous surface. The composite membranes resulted in less post-operative adhesion of the peritoneum and cecum than shown by non-treated rats without physical barriers, which could be used as a physical barrier to prevent abdominal post-operative adhesion during healing.