2A Sequences and Polycistronic Vector Design Figure 1. The construction of 2A polycistronic expression cassettes. (A) All 2A polycistronic expression cassettes were generated by restriction enzyme digestion and ligation utilising the restriction enzyme cutting sites: SacI, NotI, BamHI, HindIII and SalI (boxed, as described in Materials an methods) present in the multicloning site of the pCMV-Script plasmid (Stratagene). (B) The amplification of Ngn3-F2A, Pdx1-E2A and Nkx2.2-T2A, a size difference between the native full-length gene and the gene with a 2A-peptide tag can be observed using gel electrophoresis. Note that the native Nkx2.2 amplified from MIN6 cell cDNA contained regions outside of the coding region of the mRNA, as the primers were optimised for maximum efficiency, and thus is larger in size compared to Nkx2.2-T2A. Each group of three lanes is (from left to right) molecular weight ladder, native sequence, and native sequence +2A. (C) Examples of the flexibility of the restriction enzyme digestion/ligation construction method. Each PLOS ONE | www.plosone.org 3 November 2012 | Volume 7 | Issue 11 | e48668