cHS4 Insulation of Transposon-Delivered Transgenes Figure 2. Silencing of SB, PB, and Tol2 transposon-based vectors in ARPE-19 cells. (A) Percentage of retained median fluorescence intensity (MFI) for stably transfected ARPE-19 clones. ARPE-19 cells were transfected with pSBT/RGIP, pPBT/RGIP, or pTol2T/RGIP together with a transposase expressing plasmid, and selected for puromycin resistance. Individual clones were expanded and then passaged for 8 weeks in the absence of selection. Their eGFP expression level was determined at day 0 and day 56 of passage, and their percentage of retained MFI was calculated. (B) Percentage of retained median fluorescence intensity (MFI) for stably transfected ARPE-19 clones containing 1–3 transposon insertions. (C) Percentage of retained median fluorescence intensity (MFI) for stably transfected ARPE-19 clones containing 9 or more transposon insertions. (D) Reactivation of eGFP expression by TSA treatment. A subset of silenced ARPE-19 cell clones was grown in the presence of the deacetylase inhibitor Trichostatin A (TSA). The clones were treated 24 hours before analysis of eGFP expression by flow cytometry. doi:10.1371/journal.pone.0048421.g002 uninsulated clones, the result suggests that incorporation of cHS4 sequences into the SB and PB vectors could lead to increased gene expression levels from the transgene cassette. Similarly, insulated SB and PB clones containing 1 to 3 transposon insertions exhibited between 1.4 and 2.3-fold increased expression levels (Figure 4C). However, the positive effect of the cHS4 insulator on transgene PLOS ONE | www.plosone.org 5 October 2012 | Volume 7 | Issue 10 | e48421