transposon family, has high transposition activity in numerous invertebrate organisms [21], and is highly active in mouse and human cells [22,23,24], including therapeutically relevant cells such as human embryonic stem cells [25] and human primary T cells [26,27,28,29]. The PB transposon has also been shown to be a suitable vector system for reprogramming of human and mouse fibroblasts to induced pluripotent stem cells [30]. The Tol2 element, a member of the hAT transposon family, is the favored transposon system for transgenesis and insertional mutagenesis studies in zebrafish ([31]). In addition, the Tol2 transposon system has been shown to be active, although with lower efficiency than SB and PB, in all vertebrate cells tested so far [32]. The SB, PB and Tol2 transposons originate from different phylogenetic backgrounds and possess different biological proper- ties that may represent strengths or weaknesses in a vector context. Understanding functional differences contributes to an improved basis for choosing the most suited transposon vector system for a particular experimental or therapeutic application. Such differ- ences include, first of all, the way of transposition. Excision of the SB transposon results in 2 or 3 bp 39-overhangs at the transposon ends, and repair of the 39-overhang creates, together with the TA target site duplication, a characteristic transposition footprint [33]. Excision of PB, in contrast, results in hairpin formations at the excised transposon ends, and the 59-TTAA overhangs created in the flanking DNA after excision anneal in the absence of DNA synthesis, leaving an intact excision site without any transposition footprint [34]. Tol2 also forms hairpin structures during transpo- sition, but the hairpins are formed at the ends of the flanking donor DNA instead of at the ends of the excised element, and footprints of variable lengths are created at the excision site [35]. Secondly, SB, PB and Tol2 differ in their cargo-capacity. Whereas SB vectors have been observed to have a reduced transposition activity with increased transposon size [36,37], PB and Tol2 seem less affected by cargo size and may carry up to at least 10-kb sequences without a significant reduction in transposition activity [24,38]. Notably, the PB transposase was recently reported to mobilize a 100-kb transposon in mouse embryonic stem cells [39]. Thirdly, the integration site preferences of the three transposon systems vary. At the primary DNA sequence level, the SB and PB transposons are very strict in their choice of target site (TA dinucleotide or TTAA tetranucleotide, respectively), whereas the Tol2 transposon is less discriminating by targeting typically an 8-bp sequence that can vary in nucleotide composition. At the genomic level, SB has a fairly random integration profile with no preference for or against genes [40,41,42,43]. PB, in contrast, exhibits a nonrandom integration pattern with about 50% integration events within intragenic regions (dependent on the cell type) and a bias towards transcriptional start sites [23,28,42,43,44] and actively transcribed genes [28]. In case of Tol2, the integration pattern seems to vary depending on cell type with reports of less than 40% to almost 50% of the insertion events within transcriptional units and with a preference for integration near transcriptional start sites [43,44,45,46]. Transcriptional repression of transgene cassettes constitutes a potential problem for transgenesis and therapeutic gene transfer applications. Recombinant retroviruses are highly susceptible to transcriptional silencing and position effects conducted by chromosomal sequences at the integration sites [47,48,49]. We have previously detected transcriptional silencing of a SB transposon-delivered transgene cassette in HeLa and F9 murine teratocarcinoma cells [50,51]. By analyzing gene expression from F9 clones harboring SB transposons encoding a reporter gene driven by an RSV promoter, we observed that approximately 50% of the clones were silenced after 7 weeks of passage under non- cHS4 Insulation of Transposon-Delivered Transgenes selective conditions. Thus, SB does not seem to contain natural insulator elements that protect its content from pathways of transcriptional silencing. Such insulators are cis-acting elements that can block enhancer-activation of a promoter and/or protect transcribed regions from chromosomal position effects, offering protection against spreading of condensed inactive chromatin. The 59-HS4 chicken b-globin (cHS4) insulator, a 1.2-kb sequence located at the 59 end of the chicken b-globin domain, possesses both enhancer- and barrier-blocking effects, the latter which are coupled to hyperacetylation of histones H3 and H4 as well as a reduction in promoter CpG methylation [52]. Incorporation of cHS4 sequences into retroviral vectors improves expression of integrated transgene cassettes [53,54] and provides increased and consistent expression of therapeutic transgenes delivered by lentiviral vectors [55]. The incorporation of cHS4 sequences into SB DNA transposon vectors also has positive influence on the stability of transgene expression in embryonic cells [51]. Cells of the retina pigment epithelium (RPE) are target cells of ongoing gene therapy trials [56,57]. A spontaneously arising human RPE cell line, ARPE-19, has functional properties of RPE cells in vivo [58] and has been used frequently as an in vitro platform for molecular and genetic studies of retinal pigment epithelium. Recently, genetically engineered ARPE-19 cells were explored as a cellular source of nerve growth factor (NGF). ARPE-19 cells were encapsulated in a biodelivery device that was implanted in the basal forebrain of minipigs, facilitating in vivo delivery of NGF [59]. The therapeutic potential of this approach was recently further supported by the production of an improved ARPE-19-derived cell line in which NGF was expressed from a total of four integrated SB DNA transposon vectors encoding NGF [60]. In the present study, we further investigated long-term expression of DNA transposon-directed transgene expression in ARPE-19 cells with the goal of investigating transcriptional repression in these cells and compare cHS4-mediated insulation of expression cassettes contained within SB, PB, and Tol2 transposon vectors. By analyzing eGFP expression driven by the silencing-prone RSV promoter in stably transfected ARPE-19 cell clones, we observed progressive transgene silencing as expected but notably detected variable levels of transgene silencing for the three transposon systems. Under these conditions, the PB system was least affected by transcriptional repression. Although cHS4 insulator sequences flanking the transgene cassette were found to protect against early position effects, resulting in increased initial transgene expression, our data also demonstrate that cHS4 elements do not offer long- term protection against repression in ARPE-19 cells, and shut- down of expression was seen even in clones with multiple transposon insertions. Interestingly, our findings demonstrate that incorporation of cHS4 sequences is beneficial for mobilizing SB transposons from plasmid DNA supporting an improved stable transfection rate. Results Efficient SB, PB and Tol2 transposition in ARPE-19 cells To study mobilization and transgene expression levels of DNA transposon-derived vectors in ARPE-19 cells, we generated vectors in the context of SB, PB, and Tol2, containing an RSV-driven bicistronic gene cassette consisting of the eGFP reporter gene, an internal ribosomal entry site (IRES), and the puromycin resistance gene (puro) [51]. The vectors were designated pSBT/RGIP, pPBT/RGIP, and pTol2T/RGIP, respectively (Figure 1A). Transposition efficiencies of the three vectors in ARPE-19 cells were measured by standard colony formation assays. Equal molar amounts of pSBT/RGIP, pPBT/RGIP, and pTol2T/RGIP were PLOS ONE | www.plosone.org 2 October 2012 | Volume 7 | Issue 10 | e48421