2A Sequences and Polycistronic Vector Design Figure 7. The effect of a different promoter on the beneficial effect of T2A on EGFP expression in four-gene constructs. A significant increase (P,0.05) in mean EGFP expression per positive cells can be observed when comparing a four-gene construct linked with only T2A peptides and a four-gene construct linked with a mixture of E2A, T2A and F2A peptides, irrespective of promoter. The CAG promoter in place of the CMV promoter increased expression of EGFP, irrespective of the 2A sequences (p,0.01). There was a significant interaction effect between 2A sequence and promoter (p,0.05), showing that CAG together with al T2A gave the highest level of expression. Data presented as mean + SEM from 3 independent experiments. AU = arbitrary units. doi:10.1371/journal.pone.0048668.g007 genes linked by T2A. Thus, Pdx1-T2A and Ngn3-T2A were used to directly replace Pdx1-E2A and Ngn3-F2A in the original cassettes. The expression of these new vectors in ES cells showed that in otherwise identical two-gene cassettes the use of a T2A-linker increased expression compared to F2A or E2A (Figure 6A). Using this same approach to reconstruction of the cassette in three-gene constructs, replacing only one 2A peptide (E2A or F2A) with T2A did not result in a significant change in EGFP expression, but exclusive use of T2A in this three-gene vector resulted in an approximate 60% increase in EGFP expression (Figure 6B). CMV is not always an efficient promoter in ES cells and it was therefore of interest to determine whether the increased efficiency of T2A was also manifested when the construct was under a stronger promoter. We re-designed four-gene cassettes to be expressed under either CMV or the CAG enhancer/promoter. Constructs were designed to have either different 2A sequences between each gene or linked exclusively by T2A sequences. EGFP expression was significantly higher under the CAG promoter than CMV vectors, irrespective of the 2A sequences used (including those linked exclusively with T2A) (Figure 7). The result shows that the beneficial effect of T2A sequence on expression was independent of the efficiency of the promoter used. Discussion The present study constructed various polycistronic expression cassettes by linking a number of Gene-2A sequences, each flanked by unique restriction enzyme sites. This method allowed the generation of a combinatorial array of constructs containing up to four genes. This provides for a more time and cost-effective approach to the construction of multiple constructs and provides for a rapid method of their re-engineering as required [24]. Additionally, this method allowed the use of multiple copies of the same 2A peptide within an expression cassette without the need for use of sequence variants [30]. This eliminated any potential changes in expression or cleavage efficiency caused by using sequence variants of the same peptide [22], and therefore reduces the level of validation of the constructs required. This study confirms several reports that the efficiency of transcription decreases with an increase in the number of genes contained in a 2A polycistronic vector [7,8]. We found that a significant component of this loss of efficiency could be ameliorated by changing the identity of the 2A sequence used. Expression cassettes containing the T2A sequence were tran- scribed more efficiently than those containing either F2A or E2A in mouse ES cells, and four-gene cassettes linked exclusively with PLOS ONE | www.plosone.org 11 November 2012 | Volume 7 | Issue 11 | e48668