average copy number was comparable for all three groups, with 6.3 copies per clone for SB clones, 5.9 copies per clone for PB clones and 6.1 copies per clone for Tol2 clones. Hence, the small difference in transposition activity obtained with the three transposase variants did not translate into a substantial difference in the transposon copy number. Postintegrative silencing of RGIP-containing ARPE-19 clones – the PB vector seems less vulnerable to transcriptional repression compared to SB and Tol2 To compare the persistency of gene expression from genomi- cally inserted SB, PB and Tol2 vectors, the isolated clones were passaged for 8 weeks in the absence of puromycin, and eGFP expression levels were measured by flow cytometry at day 0 and day 56 of passage. Expression profile overlays are shown in Supplementary Figures S1 to S3. A reduction in transgene expression after 56 days of passage was observed for cell clones derived from all three transposon systems, but the level of reduction varied between the three vectors. For PB vector insertions, 33% of the clones had lost more than 50 percent of their initial expression, whereas 45% and 55% of the clones containing Tol2 and SB transposons, respectively, had lost more than half of the initial expression (Figure 2A). A statistical comparison of SB and PB datasets, by the nonparametric Mood’s median test, revealed a p-value of 0.061, showing that the decreased level of transgene repression observed for clones carrying the PB vector compared to clones containing the SB vector, was close to being statistical significant. When considering the clones with a low transposon copy number ranging from 1 to 3 copies per clone, SB and Tol2 showed comparable levels of transgene silencing with 67% of the SB clones and 64% of the Tol2 clones having lost more than 50 percent of their initial eGFP expression (Figure 2B). For the set of PB clones, 42%, of the clones expressed less than half of the initial eGFP expression, suggesting again that PB insertions carrying the RSV-driven gene cassette were less vulnerable to silencing. Notably, even ARPE-19 clones containing 9 or more transposon insertions were transcrip- tionally silenced over time (Figure 2C). Of the 8 SB clones containing 9 or more transposon copies, 38% had lost more than 70 percent of their initial transgene expression, and half of the clones had lost more than 50 percent of their initial expression. Of the 7 PB clones, 42% had lost more than half of their initial expression, but none of these clones had lost more than 70 percent of their initial expression. Notably, Tol2 clones with 9 or more transposon copies had a modest reduction in transgene expression, with all 7 clones keeping more than 50% of their initial eGFP expression, indicating that expression was retained by Tol2 in high copy clones. To verify that the reduction in transgene expression observed for each transposon system was a result of epigenetic mechanisms and not a consequence of other factors such as loss of genetic material, we treated a subset of clones from each group with the histone deacetylase inhibitor TSA. Flow cytometric analysis 24 hours after TSA treatment showed that addition of TSA could fully or partially restore transgene expression levels for all treated clones (Figure 2D). Together our data indicate that genomically integrated RSV-driven eGFP transgene cassettes were subjected to transcriptional silencing in the context of all three transposon vector systems. This is expected, as the RSV promoter is known to be prone to transcriptional silencing. Nevertheless, the degree of transcriptional repression varied between the systems, suggesting that the PB system in the context of ARPE-19 cells was overall less vulnerable to transcriptional repression. cHS4 Insulation of Transposon-Delivered Transgenes Increased transgene expression levels from insulated SB and PB transposon vectors, but limited long-term protection against silencing by cHS4 insulators in ARPE- 19 cells We have previously observed that cHS4 insulators flanking the RSV-GIP transgene cassette in the context of an SB vector leads to protection of the genomically inserted transgene against silencing in embryonic carcinoma cells [51]. To investigate the effect of cHS4 insulators in all three vector systems, we inserted two transgene-flanking cHS4 sequences in each of the vectors resulting in the plasmids pSBT/cHS4.RGIP.cHS4, pPBT/ cHS4.RGIP.cHS4, and pTol2T/cHS4.RGIP.cHS4 (Figure 3A). The stable transfection rate of the insulated transposon vectors in ARPE-19 cells was analyzed by colony formation assays. As shown in Figure 3B, a significant 1.3-fold increase in the number of puromycin-resistant colonies was observed for transfections with SBT/cHS4.RGIP.cHS4 relative to transfections with SBT/RGIP (p = 0.0079) despite the increased size of the transposon vector. A similar positive effect of the cHS4 insulator on the stable transfection rate could not be observed for PB and Tol2 vectors (Figure 3B). To generate ARPE-19 clones with insulated transposon vectors, groups of ARPE-19 clones containing SBT/ cHS4.RGIP.cHS4 (total of 24 clones), PBT/cHS4.RGIP.cHS4 (total of 28 clones), and Tol2T/cHS4.RGIP.cHS4 (total of 24 clones), respectively, were isolated and expanded. The average number of transposon insertions (determined by Southern blot analysis; not shown) was 5.5, 5.3, and 5.6 copies per clone in the three cHS4-insulated transposon groups, respectively (Figure 3C), indicating that the insulated transposon vectors gave rise to a slightly lower average transposon copy number compared to uninsulated vectors. To study possible protective effects of the cHS4 element in SB, PB and Tol2 vectors in ARPE-19 cells, clones containing insulated transposon vectors were passaged for 8 weeks under non-selective conditions. The eGFP expression level was measured for each clone by flow cytometry at day 0 and day 56 of passage (Supplementary Figures S4 to S6). Notably, we did not observe a long-term protective effect of the cHS4 element against transcriptional silencing of the RSV-GIP transgene cassette (Figure 4A). Rather, a large portion of the clones carrying insulated transposon vectors were subjected to transcriptional repression. Insulated Tol2 clones were most affected, with 79% of the clones having lost more than 50 percent of their initial expression compared to 58% and 57% for SB- and PB-containing clones, respectively (Figure 4A). TSA treatment of a subset of clones carrying insulated vectors showed that expression levels could be restored for all treated clones (data not shown), indicating again that epigenetic modifications were responsible for the decrease in transgene expression. To determine if the cHS4 insulator had an effect on transgene expression levels, we examined the median fluorescence intensity (MFI) level for clones with and without cHS4 sequences. In each group of ARPE-19 clones, large variations in MFI were observed, even for clones with an equal copy number (Supplementary Figure S7), suggesting that the chromosomal environment at the insertion site had a substantial impact on eGFP expression due to position effects. However, the mean values of MFI were increased 2.2-fold and 1.5-fold for clones carrying insulated SB and PB vectors, respectively, compared to clones with uninsulated vectors at day 0 of passage (Figure 4B). At day 56 of passage, this relative increase in mean MFI was 1.9-fold for insulated SB clones and 1.3-fold for insulated PB clones. Since the average transposon copy number was slightly lower for insulated clones compared to PLOS ONE | www.plosone.org 4 October 2012 | Volume 7 | Issue 10 | e48421