1. PiggyBac Transposon-mediated Gene Transfer in Human Cells
Matthew H Wilson, Craig J Coates, Alfred L George 
Molecular Therapy 
Volume 15, Issue 1, Pages (January 2007)
DOI: /sj.mt
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
PiggyBac exhibits efficient transposition in human cells. (a) Schematic of transposase and transposon constructs. CMV, immediate early CMV promoter; intron, SV40 intron; pA, polyadenylation sequence; SB12, hyperactive SB transposase; pT3, hyperactive SB transposon with identical IR elements; Kan/Neo, kanamycin/neomycin resistance cassette; p15A, origin of replication; SB IR elements are shaded; piggyBac IR elements are hatched. (b, c) Transposition assays comparing SB12 with piggyBac (N=3±SEM). HEK-293 or HeLa cells were transfected with transposase (400 ng) and transposon (2 μg) plasmids, passaged into G418-containing media, and selected for 2 weeks as described in Materials and Methods. *P<0.05 by two-way analysis of variance comparing piggyBac transposition with that of SB12. #P<0.05 by two-way analysis of variance comparing HEK-293 cells with HeLa cells for the given transposase.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
Excision assay of SB and piggyBac. Three days after HEK-293 cells were transfected, plasmid DNA was isolated and used as a template for PCR to amplify from plasmids that have undergone excision of the transposon segment and repair (representative data from one of three experiments are illustrated).
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
Sequence logo analysis of piggyBac and SB integration sites. WebLogo was used to analyze known piggyBac integration sites for possible consensus target sites for integration. Shown are the (a) determined consensus logo and (b) frequency plots from integration sites determined as described herein.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
PiggyBac lacks overproduction inhibition. The presence or absence of overproduction inhibition of piggyBac (with pTpB) and SB12 (with pT3) was evaluated at (a) 2 μg, (b) 200 ng, and (c) 50 ng of transposon DNA with increasing the amount of transposase transfected in HEK-293 cells (N=3±SEM). DNA was kept constant throughout all transfections using non-recombinant pIRESpuro3 plasmid. (d) The maximal activity of piggyBac was compared with that of SB12 at the varying transposon DNA amounts (N=3±SEM). (e) Western analysis of SB12 and HA-piggyBac illustrating increased transposase expression with increased transfected transposase DNA (representative data from one of three experiments). Cells were transfected with equivalent DNA amounts exactly as in the overproduction inhibition assays. Each lane was loaded with 15 μg of protein lysate. *P<0.05 comparing piggyBac with SB12 at the transfected transposase DNA amount (a–c) or maximal activity at the given transposon DNA amount (d).
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

6. Figure 5
A helper-independent piggyBac transposase–transposon with enhanced activity in human cells. (a) Schematic of helper-independent vectors with components as described in Figure 1a. (b) Transposition assays of helper-independent vectors in HEK-293 cells. Shaded bars represent transfections with the transposase (1 μg) and transposon (1 μg) supplied separately on two different plasmids. Open bars represent transfections with helper-independent vectors (1 μg of helper independent vector with 1 μg of pIRESpuro3 to keep DNA amount constant). N=3±SEM. *P<0.05 comparing PB+pTpB with SB12+pT3. **P<0.05 comparing pPB-Nori with PB+pTpB. Statistical analysis was performed using analysis of variance followed by a Bonferroni post-test comparison.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions