2A Sequences and Polycistronic Vector Design Data was obtained from 3 independent experiments. (C) The relationship between EGFP expression per cell and number of genes within constructs. An inverse linear relationship between the number of genes contained within each vector and the mean EGFP expression levels per positive cell was observed, within an increased number of genes associated with decreased EGFP expression levels. R2 = linear regression coefficient. AU = arbitrary units. doi:10.1371/journal.pone.0048668.g003 Figure 4. The expression of a gene at different locations within a 2A polycistronic expression cassette. (A) eGFP-F2A was amplified using PCR, and the difference in size compared to the native eGFP can be observed using gel electrophoresis. (B)The expression of EGFP was assessed when it was moved from the final position to the first position withinin a four-gene polycistronic construct. No statistical difference in EGFP expression was found (left axis), and no difference was found in the proportion of the cells expressing EGFP 24 hr after the cells were transfected with the two constructs (right axis). Data presented as mean + SEM from 3 independent experiments. AU = arbitrary units. doi:10.1371/journal.pone.0048668.g004 PLOS ONE | www.plosone.org 7 November 2012 | Volume 7 | Issue 11 | e48668