cHS4 Insulation of Transposon-Delivered Transgenes Figure 3. Transposition of insulated SB, PB, and Tol2 transposon vectors in ARPE-19 cells. (A) Schematic representation of pSBT/ cHS4.RGIP.cHS4, pPBT/cHS4.RGIP.cHS4, and pTol2T/cHS4.RGIP.cHS4 vectors. IR, inverted repeat; cHS4, 59-HS4 chicken b-globin insulator sequence; RSV, Rous sarcoma virus promoter; eGFP, enhanced green fluorescent protein; IRES, internal ribosome entry site; puro, puromycin resistance gene; pA, polyadenylation site. (B) Stable transfection rates of SB, PB, and Tol2 transposon vectors in ARPE-19 cells. 0.125 pmol transposon plasmid was transfected together with 0.05 mg transposase plasmid or pcDNA3.1D/V5.TOPO plasmid (empty vector) into ARPE-19 cells. The pcDNA3.1D/V5.TOPO plasmid was also included as non-specific DNA to ensure that the total amount of DNA was 1 mg for each transfection. After 8 days of selection, puromycin-resistant colonies were stained and counted. Mean 6 SEM values are shown (N = 3). P values listed above the brackets were obtained by student’s t-tests. (C) Transposon copy number of stably transfected ARPE-19 clones containing insulated transposon vectors. Genomic DNA from ARPE-19 cell clones carrying SBT/cHS4.RGIP.cHS4, PBT/cHS4.RGIP.cHS4, or Tol2T/cHS4.RGIP.cHS4 transposons was purified and examined by Southern blot analysis to determine the transposon copy number. doi:10.1371/journal.pone.0048421.g003 expression levels was most pronounced in clones with a high copy number, where the increase in mean MFI was observed to be as high as 3.7-fold for SB clones and 2.7-fold for PB clones at day 56 of passage (Figure 4D). In Tol2 clones, mean MFI values were consistently lower for clones carrying insulated transposons compared to clones containing uninsulated transposons, suggesting that incorporation of cHS4 sequences into the Tol2 vector did not lead to a protection against repressive position effects. In summary, these results indicate that the cHS4 insulator did not have a long-term stabilizing effect on transgene expression from SB, PB, and Tol2 vectors in ARPE-19 cells, but that inclusion of cHS4 sequences had an overall beneficial effect on the level of transgene expression from integrated SB and PB transposon vectors. PLOS ONE | www.plosone.org 6 October 2012 | Volume 7 | Issue 10 | e48421