genotoxicity by 6-fold in a murine tumor transplantation model [75]. The enhancer blocking activity of the cHS4 insulator was not addressed in this study, but previous analysis of promoter activation by an SV40-neo transgene unit within an SB transposon vector showed that the cHS4 element could effectively block transactivation of a nearby TATA-box minimal promoter in HeLa and primary human T cells [76]. Incorporation of cHS4 sequences could therefore potentially lead to an increased safety profile. By following the expression of a silencing-prone promoter, we demonstrate in this study that the extent of silencing may depend on the type of carrier, most likely due to overall differences in the integration profile of the different DNA transposon carriers. We show that incorporation of cHS4 insulator sequences can lead to an increase in transgene expression levels for genomically integrated SB- and PB-based vectors in ARPE19 cells. In addition, improved stable transfection rates are obtained for cHS4-insulated SB vectors, possibly due to the increased mobilization of cHS4-containing transposons from plasmid DNA. Finally, we find that inclusion of cHS4 elements in SB-, PB- and Tol2-derived vectors does not lead to long-term protection against progressive transgene silencing in ARPE19 cells, supporting the notion that the barrier activity of the cHS4 insulator is not uniformly active in all cell types. Materials and Methods Plasmid construction The plasmids pSBT/RGIP, pSBT/cHS4.RGIP.cHS4, and pCMV-SB100X have been described previously [51,77]. The pPBT/RGIP and pTol2T/RGIP plasmids were constructed by ligation of a RSV.eGFP.IRES.puro PCR fragment, amplified from pSBT/RGIP, into ClaI/NotI-digested pXL-BacII [78] and NheI/ClaI-digested pT2AL200R150 [20], respectively. To gen- erate pPBT/cHS4.RGIP.cHS4 and pTol2T/cHS4.RGIP.cHS4, the 1200-bp cHS4 insulator element was amplified from pSBT/ cHS4.RGIP.cHS4 by PCR and inserted in front of and after the RSV.eGFP.IRES.puro cassette in pPBT/RGIP (using a ClaI and NotI site) and in pTol2T/RGIP (using a NheI and ClaI site), respectively. The pCMV-PB and pCMV-Tol2 plasmids have been previously described in [79]. The pCMV-SB100X.chloramp and pCMV-PB.chloramp plasmids were generated by ligation of a chloramphenicol PCR fragment amplified from pBC SK+ (Stratagene, Santa Clara, CA) into PvuI-digested pCMV- SB100X and pCMV-PB, respectively. To generate pPBT4tp, the transposon sequence of pPBT/RGIP was cut out by NsiI/PstI- digestion, and the digested plasmid backbone was then exposed to T4 DNA polymerase treatment and blunt-end ligation. All produced DNA constructs were verified by restriction digestion and DNA sequencing. Cell culture and transposition assays HeLa (human cervical cancer) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum (Lonza, Basel, Switzerland), 0.26 mg/ml glutamine, 54 ng/ml penicillin and 36 ug/ml streptomycin. ARPE-19 human retinal pigment epithelium cells [58] were maintained in culture medium containing 50% Ham’s F-12 Nutrient Mixture (Invitrogen, Carlsbad, CA) and 50% DMEM with serum, glutamine, penicillin, and streptomycin, as described above. To measure rates of stable transfection, cells were plated at 1.5 x 105 cells/well in 6-well dishes 1 day before cotransfection with 0.125 pmol transposon plasmid and 0.016 pmol transposase plasmid or pcDNA3.1D/ V5.TOPO plasmid (Invitrogen, Carlsbad, CA) as a negative control. The pcDNA3.1D/V5.TOPO plasmid was used as stuffer cHS4 Insulation of Transposon-Delivered Transgenes DNA to obtain equal DNA amounts in each transfection. Transfections were carried out using FuGene-6 (Roche, Basel, Switzerland) according to manufacturer’s instructions using 3 ml of reagent per 1 mg of DNA. One day after transfection, cells were split in varying densities and plated in 10-cm dishes. Two days after transfection, selection medium containing 1 mg/ml puromy- cin (Invitrogen, Carlsbad, CA) was added to the cells. After 8 days of selection, colonies of cells were stained with 0.6% methylene blue, air-dried and counted. Generation of stable expressing cell clones and long- term expression analysis ARPE-19 cells were seeded in 6-well dishes (1.56105 cells/well) and transfected with 0.125 pmol transposon plasmid together with 0.05 mg transposase plasmid using FuGene-6 transfection reagent according to manufacturer’s instructions. One day after transfec- tion cells were split in varying densities and plated in 10-cm dishes. Selection medium, containing 1 mg/ml puromycin, was added to the cells two days after transfection. After 10 days of selection, single clones were isolated and expanded for genomic DNA extraction and long-term eGFP expression analysis. The isolated, stably expressing cell clones were passaged for 8 weeks in standard culture medium, and analyzed by flow cytometry on day 0 and day 56 on a BD FACSAria III cell sorter (BD Biosciences, San Jose, CA). In the flow cytometric analysis, non-transfected cells were included as a negative control, and propidium iodide (Sigma Aldrich, St Louis, MO) was used to exclude non-viable cells. Transposon excision assay HeLa or ARPE-19 cells were seeded in 6-well dishes (1.56105 cells/well) and transfected with 0.25 pmol (HeLa) or 0.125 pmol (ARPE-19) transposon plasmid together with 0.25 pmol (HeLa) or 0.05 mg (ARPE-19) transposase plasmid or pBC SK+ plasmid as negative control. The pBC SK+ plasmid was also used as stuffer DNA. Transfections were carried out using FuGene-6 according to manufacturer’s instructions using 3 ml of reagent per 1 mg of DNA. Low-molecular weight DNA was extracted 2 days after transfection using the QIAprep miniprep kit (CA 91355; Qiagen, Valencia, Spain) according to the manufacturer’s instructions, except for a 1-h incubation period at 55uC (0.6% SDS and 0.08 mg/ml proteinase K together with buffer P1) instead of the lysis step with buffer P2 after resuspension of the cell pellet. 50 ng of extracted DNA from each transfection was used in a quantitative PCR analysis using a primer set recognizing transposon excision circle products and a primer set amplifying an amplicon within the Amp resistance gene. The plasmids pUC19 and pPBT4tp were used as templates for standard curve formation. The analysis was performed on a Lightcycler 480 (Roche, Basel, Switzerland) using the Lightcycler DNA Master SYBR Green I kit (Roche, Basel, Switzerland). Amplification was performed under the following conditions: Thirty cycles (95uC 30s, 65uC 30s, and 72uC 5s). Primer sequences are as follows: SB excision (S): 59-CGAT- TAAGTTGGGTAACGCCAGGG-39, SB excision (AS): 59- CAGCTGGCACGACAGGTTTCCCG-39, PB excision (S): 59- CCGTGGAGGACGGGCAGACTCGCG-39, PB excision (AS): 59-GGCGTGCATGGCCACACCTTCCCG-39, Amp (S): 59- CAAGAGCAACTCGGTCGC-39, Amp (AS): 59- TCGTTGTCAGAAGTAAGTTGGC-39. Southern Blot analysis Genomic DNA was prepared from cell pellets following NaCl extraction and ethanol precipitation. 15 mg genomic DNA was digested with PstI+KpnI (pSBT/RGIP stable clones), KpnI+SalI PLOS ONE | www.plosone.org 10 October 2012 | Volume 7 | Issue 10 | e48421