2A Sequences and Polycistronic Vector Design Figure 2. Analysis of expression and cleavage of polycistronic vectors in mouse ES cells. (A) Each of the vectors shown in Table 3 were transiently transfected into mouse ES cells and EGFP expression assessed 24 h later. Representative analysis of at least 3 replicates is shown for each vector. The pCMV-Script backbone plasmid was used as a negative control and expression above these levels were taken as an evidence of positive EGFP expression. Each graph shows the relative level of EGFP expression in arbitrary units per cell against forward scatter. (B) The cleavage efficiency of E2A, T2A and F2A in mouse ES cells was determined by using western blot analysis of EGFP from protein samples extracted from cells transfected with two-gene vectors each linked by one of the three 2A peptides. doi:10.1371/journal.pone.0048668.g002 Cloning of the CMV Early Enhancer/chicken b-actin (CAG) Promoter In order to determine whether the choice of promoter influenced the efficiency of T2A, the CMV promoter was replaced by a CAG prompter/enhancer. The full CAG promoter sequence with a 5’-NheI restriction enzyme overhang was amplified with primers ASEI-CAG F and CAG NheI R (Table 1) from plasmid pCAGEN (kindly provided by Dr. C. Cepko through Addgene, plasmid number 11160) [28] using PCR. The CMV enhancer/ promoter from the pCMV-Pdx1-Nkx2.2-Ngn3-eGFP vectors was removed by digesting the vector with AseI and NheI. The CAG- NheI sequence was then digested and ligated into the AseI and NheI sites of the Pdx1-Nkx2.2-Ngn3-eGFP vectors. Fluorescence-activated Cell Sorting Transfected cells were analyzed 24 h post transfection using a BD DACS Calibur flow cytometer (BD Biosciences, Franklin Lakes, NJ). The cells were gated based on size and EGFP PLOS ONE | www.plosone.org 5 November 2012 | Volume 7 | Issue 11 | e48668