cHS4 Insulation of Transposon-Delivered Transgenes Figure 4. Insulation of SB, PB, and Tol2 transposon vectors in ARPE-19 cells. (A) Percentage of retained median fluorescence intensity (MFI) for stably transfected ARPE-19 clones carrying insulated transposon vectors. Measurements were obtained as described in Figure 2a. (B) Comparison of mean MFI levels for insulated and uninsulated clones. Stably transfected ARPE-19 cell clones were grown for 8 weeks in the absence of selection, and eGFP expression levels were measured by flow cytometry at day 0 and day 56 of passage. (C) Comparison of mean MFI levels for insulated and uninsulated clones carrying 1-3 transposon insertions. (D) Comparison of mean MFI levels for insulated and uninsulated clones carrying 9 or more transposon insertions. doi:10.1371/journal.pone.0048421.g004 Increased transposition of insulated SB transposon vectors in ARPE-19 and HeLa cells In the context of SB vectors, the stable transfection rate of cHS4-insulated vectors is higher than the uninsulated counterpart (Figure 3B and [51]). The number of resistant colonies obtained in colony formation assays is affected by several factors, including transfection efficiency, transposition activity, and expression levels of the resistance gene. To investigate in further detail, if the increase in stable transfection rate observed for the pSBT/ cHS4.RGIP.cHS4 vector was due to better transposition of the insulated element, we performed a quantitative PCR assay in ARPE-19 cells to quantify excision circles formed after transposon mobilization from plasmid DNA. A SB100X- or iPB-expressing plasmid, in which the ampicillin (Amp) resistance gene had been replaced by a chloramphenicol resistance gene, was transfected into ARPE-19 cells together with insulated or uninsulated transposon plasmid (equal molar amounts). Transfections of transposon plasmid in the absence of transposase were included as negative controls. Two days after transfection, low-molecular weight DNA was extracted, and real-time qPCR analysis was performed using a primer set flanking the transposon excision site. To account for variations in template DNA input, a PCR specific for the Amp gene in the transposon plasmid backbone was utilized to normalize the excision circle data to the amount of transposon plasmid recovered after transfection. Whereas no difference in the amount of excision circle products was observed for transfections with PBT/RGIP and PBT/cHS4.RGIP.cHS4, a 1.3-fold increase in excision circle formation was observed for transfections with SBT/cHS4.RGIP.cHS4 compared to transfections with SBT/ RGIP (Figure 5A). Although this increase was not statistically significant (p = 0.12), we reproducibly observed higher levels of excision from the SB vector harboring the insulators, suggesting that the increased stable transfection rate, obtained by flanking insulator sequences in the SB transposon vector, was partly caused by an increased level of transposon mobilization. To validate this finding in another cellular context, we investigated stable transfection rates and plasmid mobilization of SBT/RGIP and SBT/cHS4.RGIP.cHS4 transposons in HeLa cells. As shown in Figure 5B (left panel), the stable transfection rate of the insulated SB vector was also increased relative to that of the uninsulated vector in HeLa cells, with a twofold difference between the two transposon constructs (p = 0.028). In the excision assay, a 2.6-fold higher level of excision circle formation was obtained with pSBT/ cHS4.RGIP.cHS4 compared to pSBT/RGIP (p = 0.048) (Figure 5B, right panel). Collectively, our data demonstrate, that incorporation of cHS4 insulator sequences leads to an increase in the stable transfection rate of SB transposons, and that this increase is most likely caused by a beneficial effect of the cHS4 element on SB transposon mobilization from plasmid DNA as well as on persistency of transgene expression. Discussion In this study we analyzed and compared the transgene expression level from genomically inserted SB, PB, and Tol2 transposon vectors, encoding an uninsulated or cHS4-insulated RSV-driven eGFP-IRES-puro cassette, in human retinal pigment PLOS ONE | www.plosone.org 7 October 2012 | Volume 7 | Issue 10 | e48421