2A Sequences and Polycistronic Vector Design Figure 5. Analysis of vector DNA copy numbers using qPCR. Difference in EGFP expression in mouse ES cells transfected with two-gene and three-gene vectors compared to vector DNA copy numbers in the transfected cells. Vector DNA copy number was determined using qPCR and normalised to Actin. Even though significant differences in the mean EGFP expression per positive cell was observed between T2A containing constructs and non-T2A containing constructs for both two-gene (A) and three-gene (B) polycistronic vectors, the increased EGFP expression was not correlated with high vector DNA copy number. Data presented as mean 6 SEM from 3 independent experiments. AU = arbitrary units. doi:10.1371/journal.pone.0048668.g005 expression was detected at an excitation frequency of 488nm. The final data was analysed using the BD Cell Quest Pro software (BD Biosciences). Determining Vector DNA Copy Numbers In order to analyse the vector copy number in the transfected mouse ES cells, DNA from the cells was extracted using the Wizard Genomic isolation System (Promega) following the manufacturer’s instructions. An equal amount of DNA from each sample was added to each quantitative PCR (qPCR) reaction. The qPCR primer sequences for eGFP were obtained from a previous study [29]. Beta-Actin was used as a control (Table 1). All samples were then amplified using a Rotorgene RG3000 (Corbett Life Science, Concord, NSW, Australia). The Ct value of the eGFP was normalised against its corresponding b-Actin Ct value. The results are represented as the ‘‘units of eGFP DNA per unit of Actin’’. This provides a measure of relative transfection efficiency. Western Blot To investigate the cleavage efficiency of each of the 2A peptides used in the study, protein samples were extracted from an equal number of cells transfected with Pdx1-E2A-eGFP, Nkx2.2-T2A- eGFP and Ngn3-F2A-eGFP constructs. Constructs were separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked using 5% skim milk in TBS-Tween (TBST, Tris-Buffered Saline with 0.05% v/v Tween 20) before incubation with GFP primary antibody (1:5000) (Abcam, Cambridge, MA, USA) overnight at 4uC and horseradish peroxide conjugated secondary antibody (1:5000) (Abcam) for 1 h at room temperature. The samples were developed using Super- Signal West Femto Chemiluminescent Substrate (Thermo Fisher Scientific, Scoresby, VIC, Australia) and visualised using chemi- luminescence on a Fujifilm LAS-4000 (GE Healthcare, Waukesha, WI). Statistical Analysis Statistical analysis was conducted using SPSS statistics software (IBM, Armonk, NY). Students T-tests were conducted to compare the difference in EGFP expression level between each transfected sample and vector DNA copy numbers between T2A and non- T2A containing vectors. Univariate analysis was conducted to determine the effect of different construct designs containing T2A on the expression levels of EGFP when eGFP was the terminal gene within the construct. The difference between results was consid- ered significant if p,0.05. Results The present study constructed a range of polycistronic expression cassettes by linking a number of Gene-2A sequences using unique restriction enzyme sites. This contrasts with the conventional recombinant PCR method for generating 2A polycistronic expression cassettes [24]. The method made use of the multicloning site of the pCMV-Script plasmid (Figure 1A). A 2A peptide was assigned to each gene (F2A was assigned to Ngn3, E2A to Pdx1 and T2A to Nkx2.2) and amplified using conventional PCR. The size difference between the native gene and the gene with a 2A tag can be visualised using gel electrophoresis (Figure 1B). This approach allowed each component within a 2A polycistronic expression cassette to be edited by conventional restriction digestion/ligation (Figure 1C). All vectors generated in the study can be found in Table 3. Gene expression from the generated vectors was first screened using FACS detection of the expression of EGFP protein, which was incorporated as the last gene within each of the constructs. The cells transfected with the pCMV-Script backbone plasmid were used as negative controls. An equal amount of each vector was used to transfect mouse ES cells in all samples and EGFP expression was assessed 24 h post transfection by FACS analysis. All vectors generated using the restriction enzyme digestion/ ligation method resulted in the expression of EGFP in a proportion of the transfected population (Figure 2A). Western blot analysis of EGFP was performed on cells transfected with one of three different two-gene constructs (Figure 2B). It was expected that if cleavage was incomplete a chimeric protein of larger molecular mass would result. In each case, all of the EGFP detected was of the expected size, showing efficient cleavage by all three 2A sequences joined by the restriction enzyme method. These results show that each of the 2A sequences tested had equally high cleavage efficiencies and that the measurement of EGFP was a reliable proxy for assessing vector expression and 2A cleavage efficiency in this model. No relationship was found between the mean EGFP expression per positive cell and the proportion of cells positive for EGFP (Figure 3A). There was an inverse linear relationship (R2 = 0.65) between the number of genes contained within each vector and the proportion of cells that expressed detectable EGFP (Figure 3B), and a similar relationship with the level of expression in those cells that were positive for EGFP signal (R2 = 0.50) (Figure 3C). In order to investigate whether the position of a gene located in a 2A polycistronic expression cassette has an impact on its protein expression. An eGFP-F2A sequence was amplified using PCR (Figure 4A) and ligated into the first position of a four-gene expression cassette. Moving the eGFP from the last to first position within the construct did not change the level of EGFP expressed (Figure 4B), and confirms the utility of the flanking restriction enzyme approach for modifying the constructs. As well as the inverse relationship between expression and the number of genes within a construct, there was also marked variability in EGFP expression between different two and three- gene constructs. To assess whether this was accounted for by differences in transfection efficiency, the eGFP copy number (relative to b-actin) was measured by qPCR and compared to the level of EGFP expression. This showed that the level of EGFP expression did not show an obvious relationship to the gene copy number. It was observed that a construct containing the T2A peptide had the highest level of expression relative to copy number (Figure 5A, 5B). The constructs tested, however, contained a range of genes so it was not possible to determine whether the observed variability was accounted for by the efficiency of transcription/ translation of the different genes or due to the presence of different 2A sequences. We therefore used the flanking restriction enzyme approach to cassette re-design to change the constructs to have all PLOS ONE | www.plosone.org 9 November 2012 | Volume 7 | Issue 11 | e48668