(pSBT/cHS4.RGIP.cHS4 stable clones), KpnI+PvuII (pPBT/ RGIP stable clones) or KpnI (pTol2T/RGIP, pPBT/ cHS4.RGIP.cHS4, and pTol2T/cHS4.RGIP.cHS4 stable clones). Genomic DNA of non-transfected cells was used as a negative control. Genomic DNA of non-transfected cells spiked with plasmid DNA corresponding to 1 copy/cell or 3 copies/cell was used as a positive control. The digested DNA was electrophoresed in a 0.8% agarose gel and transferred to a Hybond membrane (GE Healthcare, Buckinghamshire, UK). The membrane was hybrid- ized overnight using a puro-specific [a-32P] dCTP-labelled probe. TSA treatment Silenced ARPE-19 cell clones were grown in the presence of 1200 nmol/l TSA (Sigma Aldrich, St Louis, MO). The clones were treated 24 hours before analysis of eGFP expression by flow cytometry on a BD FACSAria III cell sorter (BD Biosciences, San Jose, CA). Non-transfected cells were included as a negative control, and propidium iodide (Sigma Aldrich, St Louis, MO) was used to exclude non-viable cells. Supporting Information Figure S1 eGFP expression profiles of pSBT/RGIP clones measured by flow cytometry at day 0 and day 56 of growth in non-selection medium. (PDF) Figure S2 eGFP expression profiles of pPBT/RGIP clones measured by flow cytometry at day 0 and day 56 of growth in non-selection medium. (PDF) Figure S3 eGFP expression profiles of pTol2/RGIP clones measured by flow cytometry at day 0 and day 56 of growth in non-selection medium. (PDF) References 1. Bachmann A, Knust E (2008) The use of P-element transposons to generate transgenic flies. Methods Mol Biol 420: 61–77. 2. Moerman DG, Barstead RJ (2008) Towards a mutation in every gene in Caenorhabditis elegans. Brief Funct Genomic Proteomic 7: 195–204. 3. Ivics Z, Hackett PB, Plasterk RH, Izsvak Z (1997) Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell 91: 501–510. 4. Grabundzija I, Izsvak Z, Ivics Z (2011) Insertional engineering of chromosomes with Sleeping Beauty transposition: an overview. Methods Mol Biol 738: 69–85. 5. Ivics Z, Izsvak Z (2010) The expanding universe of transposon technologies for gene and cell engineering. Mob DNA 1: 25. 6. Xue X, Huang X, Nodland SE, Mates L, Ma L, et al. (2009) Stable gene transfer and expression in cord blood-derived CD34+ hematopoietic stem and progenitor cells by a hyperactive Sleeping Beauty transposon system. Blood 114: 1319–1330. 7. Mates L, Chuah MK, Belay E, Jerchow B, Manoj N, et al. (2009) Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nat Genet 41: 753–761. 8. Sumiyoshi T, Holt NG, Hollis RP, Ge S, Cannon PM, et al. (2009) Stable transgene expression in primitive human CD34+ hematopoietic stem/progen- itor cells, using the Sleeping Beauty transposon system. Hum Gene Ther 20: 1607–1626. 9. Jin Z, Maiti S, Huls H, Singh H, Olivares S, et al. (2011) The hyperactive Sleeping Beauty transposase SB100X improves the genetic modification of T cells to express a chimeric antigen receptor. Gene Ther. 10. Huang X, Wilber A, McIvor RS, Zhou X (2009) DNA transposons for modification of human primary T lymphocytes. Methods Mol Biol 506: 115– 126. 11. Singh H, Manuri PR, Olivares S, Dara N, Dawson MJ, et al. (2008) Redirecting specificity of T-cell populations for CD19 using the Sleeping Beauty system. Cancer Res 68: 2961–2971. 12. Huang X, Guo H, Kang J, Choi S, Zhou TC, et al. (2008) Sleeping Beauty transposon-mediated engineering of human primary T cells for therapy of CD19+ lymphoid malignancies. Mol Ther 16: 580–589. cHS4 Insulation of Transposon-Delivered Transgenes Figure S4 eGFP expression profiles of pSBT/ cHS4.RGIP.cHS4 clones measured by flow cytometry at day 0 and day 56 of growth in non-selection medium. (PDF) Figure S5 eGFP expression profiles of pPBT/ cHS4.RGIP.cHS4 clones measured by flow cytometry at day 0 and day 56 of growth in non-selection medium. (PDF) Figure S6 eGFP expression profiles of pTol2T/ cHS4.RGIP.cHS4 clones measured by flow cytometry at day 0 and day 56 of growth in non-selection medium. (PDF) Figure S7 Median fluorescence intensity (MFI) values of RPE transposon clones measured by flow cytometry at day 0 of passage. (PDF) Acknowledgments We thank the staff at the FACS core facility, The Faculty of Health Sciences, Aarhus University, for their technical assistance. We also thank Jakob Grove for statistical assistance. The authors would like to kindly acknowledge Gary Felsenfeld for providing the cHS4 insulator, Malcolm Fraser for providing piggyBac constructs, Koichi Kawakami for providing Tol2 constructs, and Lajos Ma´te´s, Zolta´n Ivics, and Zsuzanna Izsva´k for providing the plasmid encoding the SB100X transposase. Author Contributions Conceived and designed the experiments: NS JGM. Performed the experiments: NS AKH ROB NHS LDS. Analyzed the data: NS JGM. Contributed reagents/materials/analysis tools: JGM. Wrote the paper: NS JGM. 13. Wilber A, Linehan JL, Tian X, Woll PS, Morris JK, et al. (2007) Efficient and stable transgene expression in human embryonic stem cells using transposon- mediated gene transfer. Stem Cells 25: 2919–2927. 14. Orban TI, Apati A, Nemeth A, Varga N, Krizsik V, et al. (2009) Applying a ‘‘double-feature’’ promoter to identify cardiomyocytes differentiated from human embryonic stem cells following transposon-based gene delivery. Stem Cells 27: 1077–1087. 15. Aronovich EL, McIvor RS, Hackett PB (2011) The Sleeping Beauty transposon system: a non-viral vector for gene therapy. Hum Mol Genet 20: R14–20. 16. Izsvak Z, Hackett PB, Cooper LJ, Ivics Z (2010) Translating Sleeping Beauty transposition into cellular therapies: victories and challenges. Bioessays 32: 756– 767. 17. Williams DA (2008) Sleeping beauty vector system moves toward human trials in the United States. Mol Ther 16: 1515–1516. 18. Fraser MJ, Cary L, Boonvisudhi K, Wang HG (1995) Assay for movement of Lepidopteran transposon IFP2 in insect cells using a baculovirus genome as a target DNA. Virology 211: 397–407. 19. Cary LC, Goebel M, Corsaro BG, Wang HG, Rosen E, et al. (1989) Transposon mutagenesis of baculoviruses: analysis of Trichoplusia ni transposon IFP2 insertions within the FP-locus of nuclear polyhedrosis viruses. Virology 172: 156–169. 20. Kawakami K, Shima A, Kawakami N (2000) Identification of a functional transposase of the Tol2 element, an Ac-like element from the Japanese medaka fish, and its transposition in the zebrafish germ lineage. Proc Natl Acad Sci U S A 97: 11403–11408. 21. Handler AM (2002) Use of the piggyBac transposon for germ-line transforma- tion of insects. Insect Biochem Mol Biol 32: 1211–1220. 22. Wang W, Lin C, Lu D, Ning Z, Cox T, et al. (2008) Chromosomal transposition of PiggyBac in mouse embryonic stem cells. Proc Natl Acad Sci U S A 105: 9290–9295. 23. Wilson MH, Coates CJ, George AL Jr (2007) PiggyBac transposon-mediated gene transfer in human cells. Mol Ther 15: 139–145. 24. Ding S, Wu X, Li G, Han M, Zhuang Y, et al. (2005) Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice. Cell 122: 473–483. PLOS ONE | www.plosone.org 11 October 2012 | Volume 7 | Issue 10 | e48421