cHS4 Insulation of Transposon-Delivered Transgenes Figure 5. Inclusion of cHS4 sequences results in an improved SB transposon stable transfection rate. Mean 6 SEM values are shown (N = 3). P values listed above the brackets were obtained by student’s t-tests. (A) Measurement of excision circle formation after SB and PB transposition event in ARPE-19 cells. SB100X or iPB transposase expressing plasmid containing a chloramphenicol resistance gene instead of an ampicillin (Amp) resistance gene was transfected into ARPE-19 cells together with 0.125 pmol of insulated or uninsulated SB or PB transposon plasmid. Transfections of transposon plasmid in the absence of transposase expressing plasmid were included as a negative control. The pBC SK+ plasmid was included as non-specific DNA to ensure that the total amount of DNA was 1 mg for each transfection 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. (B) Stable transfection rate of insulated and uninsulated SB transposons and excision circle formation after SB transposition in HeLa cells. Equal molar amounts of pSBT/RGIP and pSBT/cHS4.RGIP.cHS4 plasmid were cotransfected together with pCMV-SB100X plasmid or pcDNA3.1D/V5.TOPO plasmid (empty vector, only included in transposition assay) into HeLa cells. The number of puromycin-resistant colonies was obtained as described in Figure 1B. doi:10.1371/journal.pone.0048421.g005 epithelium cells. By flow cytometric measurements of eGFP expression from single cell clones, we detected transcriptional silencing of the uninsulated transgene cassette in the context of all three vector systems, indicating that transcriptional transgene repression can constitute a problem in ARPE-19 cells. Previous analyses of transgene expression from SB transposon-containing embryonal carcinoma cell clones generated by a nonselective approach showed that a substantial percentage (a minimum of 37%) of the transgene insertions were subjected to complete silencing shortly after integration [51]. In the present study, transposon-containing ARPE-19 clones were generated by the use of an antibiotic selection scheme, which likely leads to a biased isolation of cell clones with transposon insertions situated in more active regions of the chromosome (as opposed to heterochroma- tin). Still, 55% of the SB clones, 45% of the Tol2 clones, and 33% of the PB clones had lost more than half of their initial eGFP expression after an 8-week period of growth under nonselective conditions. Histone deacetylation seemed to be part of the transcriptional repression of integrated transgenes, as treatment with the deacetylase inhibitor TSA was observed to regenerate transgene expression. This finding is in agreement with previous analyses of silencing of retroviral [48,62] and SB vectors [50,51]. It is likely that other epigenetic mechanisms, such as DNA methylation, were involved in postintegrative gene silencing. Indeed, we have previously observed that addition of the DNA methyltransferase inhibitor 5-Azacytidine could reactivate silenced HeLa and F9 cell clones harboring SB transposon insertions [50,51]. Extensive silencing of SB transposons carrying RSV-driven eYFP and eGFP expression cassettes has previously been observed in HeLa and F9 cells [50,51]. In contrast, low levels of transposon vector silencing was observed in a comparison study of gene expression from a Venus-IRES-neo cassette driven by the CAGGS promoter in the context of integrated SB, PB, or Tol2 transposons PLOS ONE | www.plosone.org 8 October 2012 | Volume 7 | Issue 10 | e48421