in HeLa cells [45]. As transcriptional repression of integrated vectors has been demonstrated to occur in a promoter-dependent manner [50,63], such discrepancies most likely reflect that the experiments were conducted with transposons containing different cargo sequences, suggesting that silencing of transposon-based vectors can be diminished by careful transgene design. However, substantial differences between cell types may affect the silencing profile. In a recent report examining transgene expression from SB vectors stably transfected into K562 erythroid cells, progressive transgene silencing of the CAGGS promoter was observed and could be significantly reduced by flanking the CAGGS-DsRed or IHK-b-globin gene cassettes with cHS4-insulators [64], indicating that incorporation of protective insulating elements can be beneficial, even when promoter sequences are carefully chosen. In the present study, all three transposon systems contained the same transgenic cassette, which means that potential intrinsic silencing triggers resident in the RSV promoter or the eGFP gene were present in all vectors. Nevertheless, PB vectors were less affected by transgene silencing compared to vectors based on SB and Tol2. Notably, PB has a preference for integrating into transcriptional units, whereas SB has a fairly random integration profile with no preference for or against genes [23,28,40,41,42,43]. The integration profile of Tol2 vectors seems to vary dependent on cell type with a high preference for transcriptional start sites in some cells (HeLa and primary human T cells) and a more random integration pattern in others (zebrafish and HEK293 cells) [43,44,45,46]. Although the integration profile of PB, SB, and Tol2 vectors has not been examined in ARPE-19 cells, the increased integration frequency of the PB transposon in transcrip- tionally ‘‘open’’ regions compared to SB and Tol2 transposons is an obvious explanation for the decreased level of transgene silencing observed for PB. Alternatively, the PB vector may contain protective cis-elements in its terminal regions. Analysis of the 59 and 39-terminal repeats of the PB transposon has revealed the existence of promoter activity in the 59-terminal repeat and enhancer activity in the 39-terminal repeat [65,66], but the existence of protective motifs has not been reported. In the attempt to create silencing-protected transposon vectors, we tested the effect of incorporating 1.2-kb cHS4 insulator sequences into the three vector systems, which to our knowledge is the first time the barrier function of the cHS4 element has been tested in PB and Tol2 transposon vectors. Flanking the transgene cassette with the cHS4 insulator resulted in a 2.2-fold and 1.5-fold increase in the initial eGFP expression level of SB and PB vectors, respectively, despite the fact that the average copy numbers were reduced with the cHS4-containing vectors. Similar results have been observed in studies of transgene expression from retroviral vectors in which cHS4 inclusion led to increased transgene expression levels in transduced murine fibroblast NIH3T3 cell clones [67,68], human fibrosarcoma HT1080 cell clones [53], and murine primary bone marrow progenitor clones [53]. By comparing the MFI level of stably expressing ARPE-19 clones carrying SB-, PB-, and Tol2-derived vectors, we detected large variations of transgene expression within clones containing equal transposon copy numbers, suggesting that eGFP expression levels were not simply determined by transgene copy number, but that chromosomal position effects had a large influence on eGFP expression. Since the cHS4 element does not exhibit conventional enhancer activity, the increase in transgene expression caused by the cHS4 elements likely reflects the ability of the cHS4 insulator to reduce the impact of repressive chromosomal position effects on vector transgene expression. It remains to be elucidated, however, why inclusion of cHS4 sequences into the Tol2 vector did not seem to benefit the eGFP expression level. cHS4 Insulation of Transposon-Delivered Transgenes By measuring eGFP expression levels of stably expressing ARPE-19 clones grown for 8 weeks in the absence of selection, we discovered that the cHS4 insulator did not protect against progressive transgene silencing of SB, PB, and Tol2 transposon vectors in ARPE-19 cells. Our previous eGFP expression studies of F9 cell clones harboring the SBT/RGIP or SBT/ cHS4.RGIP.cHS4 transposon vector showed that inclusion of cHS4 sequences into the SB transposon had a profound effect on the stability of transgene expression in F9 cell clones after prolonged passaging in culture. Since identical SB transposon constructs were used in this study, differences between cell types most likely accounted for the variable benefit of including cHS4 insulators. In support of this notion, Rivella et al. observed that inclusion of the cHS4 insulator into a recombinant retroviral vector could decrease vector methylation and transgene silencing in murine erythroleukemia cells, but not in murine embryonic stem cells [54], indicating that the barrier function of the cHS4 insulator is not uniformly active in all kinds of cell types. To improve the protection against progressive silencing of DNA transposon-embedded transgenes in ARPE-19 cells, the ubiqui- tously-acting chromatin opening element (UCOE) derived from the human HNRPA2B1-CBX3 locus [69] may represent an attractive alternative to the cHS4 insulator. The UCOE sequence contains a methylation-free CpG island which has been found to shield flanking heterologous promoters from transcriptional silencing, allowing sustained transgene expression from lentiviral vectors [70,71,72]. However, this element remains to be investi- gated in conjunction with a series of promoters and in the context of DNA transposon-based vectors. Previous studies have determined an inverse relationship between transposon length and transposition frequency for SB transposon vectors [36,37]. In the present study, we did not observe a reduction in transposition activity when two 1.2-kb cHS4 insulator sequences were incorporated into the pSBT/RGIP vector. Instead, significantly increased stable transfection rates were observed for the insulated SB vector in both ARPE-19 and HeLa cells. By quantitative measurements of SB excision circle formation, we detected a positive effect of the cHS4 sequences on SB transposon mobilization from plasmid DNA. The mechanisms responsible for insulator function are still poorly understood, but an ability of insulator binding proteins to form closed looped chromatin domains has been proposed as one model for insulator enhancer blocking activity [73]. The DNA-bending protein HMGBI is a cofactor of SB transposition in mammalian cells and is believed to stimulate transposition by assisting the SB transposase during synaptic complex formation either by bringing the transposon binding sites and/or the terminal repeats physically closer to each other [74]. Hypothetically, cHS4 insulator binding proteins may also stimulate DNA bending and bring the transposon binding sites closer to each other by loop formation of DNA sequences between the cHS4 element sequences, resulting in increased stabilization of the synaptic complex and increased transposition activities. This hypothesis, however, remains to be tested. Genotoxicity caused by activation of proto-oncogenes near the vector integration site constitutes a serious problem to gene therapy. As none of the SB, PB, or Tol2 transposon vectors integrate in a site-specific manner, a risk of insertional mutagenesis upon vector integration exists, a risk especially pronounced for PB and Tol2 transposons due to their increased preference for integrating into transcriptional units. The cHS4 element contains enhancer blocking activity which enables it to block molecular communication between enhancers and genes. Inclusion of the cHS4 insulator in retroviral vectors has been reported to reduce PLOS ONE | www.plosone.org 9 October 2012 | Volume 7 | Issue 10 | e48421