2A Sequences and Polycistronic Vector Design component of a 2A polycistronic expression cassette can be removed by using the appropriate restriction enzymes: (i) For Pdx1-E2A-eGFP, Pdx1-E2A can be cut out using SacI and NotI/BamHI (left), eGFP can be cut out using NotI/BamHI and SalI (middle) and Pdx1-E2A-eGFP can be cut out using SacI and SalI; (ii) for Nkx2.2-T2A-eGFP, Nkx2.2-T2A can be cut out using NotI and BamHI (left), and eGFP can be cut out using BamHI and SalI (middle), and Nkx2.2-T2A-eGFP can be cut out using NotI and SalI (right); (iii) for Ngn3-F2A-eGFP, Ngn3-F2A can be cut out using BamHI and HindIII (left), eGFP can be cut out using HindIII and SalI (middle), and Ngn3-F2A-eGFP can be cut out using BamHI and SalI (right); (iv) for Nkx2.2-T2A-Ngn3-F2A-eGFP, Nkx2.2-T2A can be cut out using NotI and BamHI (left), Ngn3-F2A can be cut out using BamHI and HindIII (middle), and eGFP can be cut out using HindIII and SalI. The sizes of the DNA fragments were confirmed by comparison to the molecular weight ladder on the left of each data set. doi:10.1371/journal.pone.0048668.g001 demonstrated the utility and efficiency of the this approach to testing polycistronic constructs. Materials and Methods Cell Cultures The mouse ES cell line D3 (P22-P32) was maintained on 0.1% (v/v) gelatin (BDH Chemicals, Poole, UK) coated tissue culture dishes in ES cell media (DMEM (Thermo Fisher Scientific, Scoresby, VIC, Australia) supplemented with 10% (v/v) FBS (Invitrogen, Carlsbad, CA), 0.1 mM b-mercaptoethanol (Sigma, St. Louis, MO), 50 mg/mL penicillin/streptomycin mix (Sigma, St. Louis, MO) and 1000 units of Leukaemia inhibitory factor (LIF) (GIBCO, Grand Island, NY)). ES cell media was changed every 1–2 days and the cells were passaged using 0.25% (w/v) trypsin-EDTA (GIBCO) upon reaching 70% confluence. Cells were cultured in 5% CO2 in air at 37uC. Amplification of the Full-length Sequences Using PCR The primers used for generating constructs, and their restriction sites are shown in (Table 1). Full-length sequences of mouse Ngn3 and Pdx1 were amplified from pAd-Ngn3-I-nGFP and pAd-Pdx1-I- nGFP plasmids, respectively (plasmid number 19410 & 19411, kindly supplied by Dr. D. Melton through Addgene, Cambridge, MA) [25] using primers Pdx1 Full F, Pdx1 Full R, Ngn3 Full F and Ngn3 Full R (Table 1). The full-length sequence of Nkx2.2 was amplified using the mouse insulinoma (MIN6) cell line [26] cDNA as a template with primers Nkx22 Full F and Nkx22 Full R (Table 1). The full-length sequence of eGFP was amplified from Table 3. List of vectors generated for this study. Vectors pCMV-eGFP pCMV-Ngn3-F2A-eGFP pCMV-Pdx1-E2A-eGFP pCMV-Nkx2.2-T2A-eGFP pCMV-Pdx1-E2A-Ngn3-F2A-eGFP pCMV-Pdx1-E2A-Nkx2.2-T2A-eGFP pCMV-Nkx2.2-T2A-Ngn3-F2A-eGFP pCMV-Ngn3-T2A-eGFP pCMV-Pdx1-T2A-eGFP pCMV-Pdx1-T2A-Ngn3-F2A-eGFP pCMV-Pdx1-E2A-Ngn3-T2A-eGFP CMV-G CMV-N-F2A-G CMV-P-E2A-G CMV-N2-T2A-G CMV-P-E2A-N-F2A-G CMV-P-E2A-N2-T2A-G CMV-N2-T2A-N-F2A-G CMV-N-T2A-G CMV-P-T2A-G CMV-P-T2A-N-F2A-G CMV-P-E2A-N-T2A-G pCMV-Pdx1-E2A-Nkx2.2-T2A-Ngn3-F2A-eGFP CMV-P-E2A-N2-T2A-N-F2A-G pCMV-eGFP-F2A-Pdx1-E2A-Nkx2.2-T2A-Ngn3 CMV-G-F2A-P-E2A-N2-T2A-N pCMV-Pdx1-T2A-Nkx2.2-T2A-Ngn3-T2A-eGFP CMV-P-T2A-N2-T2A-N-T2A-G pCAG-Pdx1-E2A-Nkx2.2-T2A-Ngn3-F2A-eGFP CAG-P-E2A-N2-T2A-N-F2A-G pCMV-Pdx1-T2A-Nkx2.2-T2A-Ngn3-T2A-eGFP CMV-P-T2A-N2-T2A-N-T2A-G doi:10.1371/journal.pone.0048668.t003 the 613bp to 1329bp of the pEGFP-C1 plasmid (Clontech, Mountain View, CA) using primers eGFP Full F and eGFP Full R (Table 1). Vector Construction The stop codons of Pdx1, Nkx2.2 and Ngn3 were removed from their respective reverse primers and a sequence encoding a glycine- serine-glycine (GSG) linker (Table 2) was added to the 3’-end of each gene. This linker is reported to facilitate efficient cleavage of the 2A peptides [27]. PCMV-Script (Stratagene, Cedar Creek, TX) was used as the backbone plasmid. Each gene was assigned a 2A peptide and flanked with appropriate restriction enzyme sites as found on the pCMV-Script plasmid. The following sequences were generated for the initial testing of the expression cassettes: SacI- Pdx1-E2A-NotI/BamHI, NotI-Nkx2.2-T2A-BamHI, BamHI-Ngn3- F2A-HindIII, BamHI/HindIII-eGFP-SalI. To move the eGFP from the final position to the first position, the F2A sequence attached to Ngn3 was removed and a stop codon was added to the end of Ngn3 to generate BamHI-Ngn3-HindIII. This replaced BamHI-Ngn3-F2A- HindIII. The stop codon of the eGFP was removed and the F2A sequence was added to the 39-end of the eGFP to become SacI- eGFP-F2A-SacI. T2A sequences were used in place of the F2A and E2A sequences, while no changes to the restriction enzyme sites used were made. The template sequences of the F2A, E2A and T2A peptides were obtained from a previously published study [11]. The primers used to generate the 2A constructs were: BamHI/HindIII-eGFP, eGFP- SalI, BamHI-Ngn3-F2A-HindIII F, BamHI-Ngn3-F2A-HindIII R, SacI-Pdx1-E2A-NotI/BamHI F, SacI-Pdx1-E2A-NotI/BamHI R, NotI-Nkx2-T2A-BamHI F, NotI-Nkx2-T2A-BamHI R, SacI-eGFP- F2A-SacI F, SacI-eGFP-F2A-SacI R (Table 1). The amplified sequences were then ligated into the multicloning site of pCMV- Script using T4 ligase (Promega, Madison, WI) to obtain the desired 2A polycistronic vectors. All restriction enzymes and buffers used were purchased from New England Biolabs (Ipswich, MA). The primers used for the T2A experiments are: BamHI-Ngn3- T2A-HindIII F, BamHI-Ngn3-T2A-HindIII R and SacI-Pdx1-E2A- NotI/BamHI F, SacI-Pdx1-T2A-NotI/BamHI R (Table 1). The generated sequences were then ligated into the desired locations of the 2A polycistronic vectors to replace the original sequences. Transient Transfection of Constructs Mouse ES cells were trypsinised and plated at 100,000 cells/ cm2 on 48 well tissue culture plates (Nunc, Roskilde, Demark). Transfection media was ES cell media without penicillin/ streptomycin. Sterile vector DNA at a concentration of 1.2 mg/ 100,000 cells was transfected using Lipofectamine 2000 at 3.6 mL/ 1.2 mg of DNA (Invitrogen). Samples were analysed 24 h post transfection. The backbone pCMV-Script plasmid (Stratagene) was used as a negative control. PLOS ONE | www.plosone.org 4 November 2012 | Volume 7 | Issue 11 | e48668