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.

Background The relationship of LPA single nucleotide polymorphisms (SNPs), apolipoprotein(a) isoforms and lipoprotein(a) [Lp(a)] levels with major adverse cardiovascular (MACE) events in different ethnic groups is not well known. Methods LPA SNPs, apolipoprotein(a) isoforms, Lp(a) and oxidized phospholipids on apolipoprotein B-100 (OxPL-apoB) levels were measured in 1792 Black, 1030 White and 597 Hispanic subjects enrolled in the Dallas Heart Study. Their interdependent relationships and prospective association with MACE after median 9.5-year follow-up were determined. Results LPA SNP rs3798220 was most prevalent in Hispanics (42.38%), rs10455872 in Whites (14.27%) and rs9457951 in Blacks (32.927%). The correlation of each of these SNPs with the major apolipoprotein(a) isoform size was highly variable and in different directions among ethnic groups. In the entire cohort, Cox regression analysis with multivariable adjustment revealed that quartiles 4 of Lp(a) and OxPL-apoB were associated with hazard ratios (HR) (95% CI) for time to MACE of 2.35 (1.50-3.69), p<0.001) and 1.89 (1.26-2.84), p=0.003), respectively, versus quartile 1. Addition of the major apolipoprotein(a) isoform and the 3 LPA SNPs to these models attenuated the risk, but significance was maintained for both Lp(a) and OxPL-apoB. Evaluating specific ethnic groups, in Blacks Lp(a) was a positive predictor and the size of the major apolipoprotein(a) isoform and inverse predictor, in Whites the size of the major apolipoprotein(a) isoform was an inverse predictor and in Hispanics OxPL-apoB was a predictor of time to MACE. Conclusion The prevalence and association of LPA SNPs with size of apolipoprotein(a) isoforms, Lp(a) and OxPL-apoB levels are highly variable and ethnicity-specific. The relationship to MACE is best explained by elevated plasma Lp(a) or OxPL-apoB levels, despite significant ethnic differences in LPA genetic markers.