2A Sequences and Polycistronic Vector Design Table 1. The sequences of primers used in this study, the restriction enzyme sites used in the study are underlined. Symbols eGFP Full F eGFP Full R SacI-GFP-F2A-SacI F SacI-GFP-F2A-SacI R BamHI/Hind-eGFP eGFP-SalI NGN3 Full F NGN3 Full R BamHI-NGN3-F2A-HindIII F BamHI-NGN3-F2A-HindIII R BamHI-NGN3-T2A-HindIII R PDX1 Full F PDX1 Full R SacI-PDX1-E2A-NotI/BamHI F SacI-PDX1-E2A-NotI/BamHI R SacI-PDX1-T2A-NotI/BamHI R NKX22 Full F NKX22 Full R NotI-NKX2-T2-BamHIA F NotI-NKX2-T2A-BamHI R ASEI-CAG F CAG-NheI R b-Actin F b-Actin R Sequences 5’-GCCACCATGGTGAGCAAG-3’ 5’-TTACTTGTACAGCTCGTCCATGC-3’ 5’-GAGCTCGCGGCCGCTGGATCCGCCACCATGGTGAGCAAG-3’ 5’-GGATCCAGCGGCCGCGAGCTCGGGCCCTGGGTTGGACTCCACGTCTCCCGCCAACTTGAGAAGGTCAAAATTCAAA GTCTGTTTCACCGGACCGCTGCCCTTGTACAGCTCGTCCATGC-3’ 5’-GGATCCAAGCTTATGGTGAGCAAGGGCGAG-3’ 5’-GTCGACTTACTTGTACAGCTCGTCCATGC-3’ 5’-ATGGCGCCTCATCCCTTG-3’ 5’-CAAGAAGTCTGAGAACACCAGTGCT-3’ 5’-GGATCCGCCACCATGGCGCCTCATCCCTTG-3’ 5’-AAGCTTGGGCCCTGGGTTGGACTCCACGTCTCCCGCCAACTTGAGAAGGTCAAAATTCAAAGTCTGTTTCACCGGA CCGCTGCCCAAGAAGTCTGAGAACACCAGTGCT-3’ 5’-AAGCTTTGGGCCAGGATTCTCCTCGACGTCACCGCATGTTAGCAGACTTCCTCTGCCCTCACCGCTGCCCAAGAA GTCTGAGAACACCAGTGCT-3’ 5’-ATGAACAGTGAGGAGCAGTACTACGCG-3’ 5’-CCGGGGTTCCTGCGGTCG-3’ 5’-GAGCTCGCCACCATGAACAGTGAGGAGCAGTACTACGCG-3’ 5’-GGATCCAGCGGCCGCAGGACCGGGGTTACTTTCAACATCGCCAGCGAGTTTCAACAAAGCGTAGTTAGTACATT GGCCAGAACCCCGGGGTTCCTGCGGTCG-3’ 5’-GGATCCAGCGGCCGCTGGGCCAGGATTCTCCTCGACGTCACCGCATGTTAGCAGACTTCCTCTGCCCTCGCCA GAACCCCGGGGTTCCTGCGGTCG-3’ 5’-CCCCAGTCACAGCCTACATT-3’ 5’-AACAACCGTGGTAAGGATCG-3’ 5’-GCGGCCGCTGCCACCATGTCGCTGACCAACACAAAGACG-3’ 5’-GGATCCTGGGCCAGGATTCTCCTCGACGTCACCGCATGTTAGCAGACTTCCTCTGCCCTCGCCAGAACCCCAA GTCCACTGCTGGGCC-3’ 5’-ATTAATAGTAATCAATTACGGGGTCA-3’ 5’-GCTAGCGAATTCTTTGCCAAAATGATGA-3’ 5’-GCAGGCCTAGTAACCGAGACA-3’ 5’-AGTTTTGGCGATGGGTGCT-3’ doi:10.1371/journal.pone.0048668.t001 systematic analysis of the effects of different 2A peptides on the protein expression levels within polycistronic expression cassette. To date, there is not a sufficient body of evidence to allow rational design of 2A-linked polycistronic constructs with optimal expres- sion efficacy. The most conventional method for generating polycistronic expression cassettes linked by 2A peptides involves the use of recombinant PCR [24]. This method is relatively inflexible because the use of the same 2A peptide sequence within a PCR Table 2. Different glycine-serine-glycine (GSG) linkers (Forward) and their corresponding reverse primer sequences (Reverse) used for the generation of 2A polycistronic constructs. Forward 5’-GGTTCTGGC-3’ 5’-GGCAGCGGT-3’ 5’-GGAAGAGGA-3’ doi:10.1371/journal.pone.0048668.t002 Reverse 5’-GCCAGAACC-3’ 5’-ACCGCTGCC-3’ 5’-TCCTCTTCC-3’ results in their self-annealing. Thus, designs are constrained by the need to use either different 2A peptides, or different variables between each linked proteins. Furthermore, any changes made to the structure of the cassette, such as the addition of a gene, requires the re-design of the large primers used in construction as well as the cost and time-consuming process of re-constructing the cassette. These matters limit the feasibility of constructing large numbers of constructs for screening and optimisation purposes. The present study assesses a more flexible approach for generating 2A polycistronic expression cassettes. Each gene and its assigned 2A peptide were designed to have flanking restriction enzyme sites. This allowed the generation of many different gene combinations by adding and removing gene-2A peptide units using standard restriction enzyme digestion and ligation methodologies. This approach allows the use of multiple copies of a given 2A peptide within an expression cassette. The aim of this study was to systematically analyze the effects of design variables on the efficiency of production and expression of 2A polycistronic vectors in mouse embryonic stem cells. The number of genes, their order within a construct, the type of promoter and the type of 2A peptide used were examined. We found that the type of promoter and the 2A peptides used independently impacted on the efficiency of expression and PLOS ONE | www.plosone.org 2 November 2012 | Volume 7 | Issue 11 | e48668