transfected together with pcDNA3.1D/V5.TOPO plasmid (empty vector) or a helper plasmid expressing either SB100X transposase [7], the native iPB transposase [24], or Tol2 transposase [61]. This set of transposase variants were not chosen with the intention of directly comparing transposition capabilities but with the expec- tation of generating series of stable cell clones with a comparable number of insertions. After 8 days of selection, puromycin-resistant colonies were stained and counted. As shown in Figure 1B, the three combinations of transposon vectors and transposase promoted variable levels of stable transfection, with the SBT/ RGIP transposon mobilized with SB100X showing a stable transfection rate that was 1.7-fold and 2.8-fold higher than the rates of PBT/RGIP (mobilized with iPB transposase) and Tol2T/ RGIP (mobilized with the Tol2 transposase), respectively (p,0.01). A more than 100-fold difference in colony formation between transfections with transposase plasmid and transfections with cHS4 Insulation of Transposon-Delivered Transgenes empty vector was observed for each transposon system, indicating that all three vectors were efficiently transposed in ARPE-19 cells. Next, we constructed three series of ARPE-19 clones containing genomic insertions of the SBT/RGIP, PBT/RGIP, or Tol2T/ RGIP transposons, respectively. Equal molar amounts of pSBT/ RGIP, pPBT/RGIP or pTol2T/RGIP were transfected together with SB100X, iPB, or Tol2 transposase-expressing plasmid, respectively, into ARPE-19 cells, and single colonies were subsequently isolated after puromycin selection. Three groups of 29 SB clones, 27 PB clones and 31 Tol2 clones were isolated in total. Southern blot analysis of genomic DNA from all expanded cell clones (representative Southern blot shown in Figure 1C) revealed a large variation in the number of transposon insertions per clone within each group. In all three groups, a variation in transposon copy number ranging from 1 copy per clone to more than 10 copies per clone was observed (Figure 1C). However, the Figure 1. Transposition of SB, PB, and Tol2 transposon vectors in ARPE-19 cells. (A) Schematic representation of pSBT/RGIP, pPBT/RGIP, and pTol2T/RGIP vectors. IR, inverted repeat; 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 of pSBT/RGIP, pPBT/RGIP, and pTol2T/RGIP plasmid were cotransfected together with 0.02 pmol pcDNA3.1D/V5.TOPO plasmid (empty vector) or 0.02 pmol helper plasmid expressing either SB100X transposase, iPB transposase, or Tol2 transposase. The pcDNA3.1D/V5.TOPO plasmid was also included as non-specific DNA to ensure that the total amount of DNA was 1 mg in 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. Genomic DNA from ARPE-19 cell clones carrying SBT/RGIP, PBT/ RGIP, or Tol2T/RGIP transposons was purified and examined by Southern blot analysis to determine the transposon copy number. A representative Southern blot is shown. doi:10.1371/journal.pone.0048421.g001 PLOS ONE | www.plosone.org 3 October 2012 | Volume 7 | Issue 10 | e48421