1. Hyperactive Transposase Mutants of the Sleeping Beauty Transposon
James Baus, Li Liu, Arnold D. Heggestad, Sonia Sanz, Bradley S. Fletcher 
Molecular Therapy 
Volume 12, Issue 6, Pages (December 2005)
DOI: /j.ymthe
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
Alignment of the integrase domain and C-terminal end of the SB10 transposase with related Tc1-like transposases. An amino acid BLAST search of the integrase domain of the SB transposase reveals a number of related proteins from divergent species. The top sequence is the original SB10 transposase, while the most homologous proteins within this area are aligned below. The GenBank accession number for each protein is shown to the left and the species sources are as follows: CAB51371 and CAC28060 are both from Pleuronectes platessa, EAA03472 and AAB02109 are from Anopheles gambiae and albimanus, respectively. CAA91458 and NP_ are derived from Caenorhabditis elegans and S60466 and AAC47095 are from Drosophila melanogaster. AAD34306 is from Haemonchus contortus. A potential consensus sequence is illustrated and served as the basis for the amino acid mutations that are contained within the N1, C1, and C2 fragments shown below the consensus. The N2 mutations are a combination of N1 plus M243Q and VVA253HVR. The numbers above the alignment refer to the amino acid number within the original SB10 transposase.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
Mutant SB transposase constructs. Shown is a graphical representation of the various transposase constructs that were generated in an effort to enhance the efficiency of transposition. (A) Diagram of fragments that comprise the HSB1–12 mutant transposase constructs. Restriction sites (HindIII) were designed between the center fragments to facilitate the cloning of the mutant HSB transposases (depicted by the long vertical lines). This region of the protein encodes the integrase domain. The small boxes show the position of and specific amino acid changes that are contained within each fragment. The fragments labeled SB10 encode amino acids identical to those portions of the original transposase. The fragment designated AAA contains the triple alanine mutations (K33A, T83A, and L91A) as well as the T136R mutation. The fragment HVR contains the mutations M243Q and VVA254HVR as described by Geurts et al. The fragments N1, N2, C1, and C2 are all described in Fig. 1. (B) Representation of the HSB mutants 13–17, which were generated to evaluate the combinatorial effect of specific hyperactive mutations. These constructs were designed to remove potential detrimental amino acid changes, such as the A250K, the I311L, and the C1 or C2 modifications.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
Transposition efficiency of the mutant SB transposases. (A) Analysis of transposase mutants HSB1–12. A neomycin-resistance transposition assay was performed in HeLa cells to assess the activity of the various mutant transposases using the transposon pT/SV-Neo. A 2:1 mass-based transposon-to-transposase ratio was used. (B) Transposition activity of the hyperactive mutants HSB13–17. A 2:1 molar ratio was used. For both graphs the data represent the means and standard deviations of at least three independent experiments performed in duplicate and are normalized to 100% based on the activity of the reference transposase pTRUF-HSB utilizing the transposon pT/SV-Neo. Above each bar is an estimate of the percentage of transfected cells that become neomycin resistant. These percentages were calculated based on a 50% transfection rate, the number of cells plated into selection medium, and a 100% plating efficiency. The asterisks demonstrate significant differences between the specific hyperactive transposases and the parental transposase HSB (two-tailed t test, P < 0.05).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
Synergy of transposition efficiency with hyperactive transposase and improved transposon. A neomycin transposition assay was performed within HeLa cells using either the pT/SV-Neo or the pMSZ-Neo transposon and the transposase indicated for each condition. The data represent the means and standard deviations of three independent experiments performed in duplicate and are normalized to 100% based on the reference construct pT/SV-Neo and the transposase pTRUF-HSB. A 2:1 transposon-to-transposase molar ratio was used for all combinations. Above each bar is the estimated percentage of transfected cells that have become neomycin resistant as described in the legend to Fig. 3. Asterisks denote significant differences between the combination of transposase and transposon used and the transposon system of pTRUF-HSB and pT/SV-Neo (two-tailed t test, P < 0.05).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

6. FIG. 5
Western analysis of transposase expression. To assess for equivalent steady-state levels of expression for the hyperactive mutants, Western analysis was performed in HeLa cells transfected with equal molar amounts of the specific transposase constructs. Equivalent protein loading was demonstrated by reprobing the blot with the constitutive protein α-actin. The large arrowhead demonstrates the major transposase band at approximately 35 kDa. In the pTRUF expression plasmids, an additional larger molecular weight transposase-immunoreactive band is observed (small arrowhead). The arrow indicates the constitutive protein α-actin. Lanes 1, control, mock-transfected cells; 2, pCMV-SB10; 3, pCMV-HSB; 4, pTRUF-HSB; 5, pTRUF-HSB11; 6, pTRUF-HSB14; 7, pTRUF-HSB17; 8, pCMV-HSB17.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

7. FIG. 6
Southern blotting of transposon integrations. HeLa cells were transfected with the transposon pT/SV-Neo and either (A) the original transposase SB10 or (B) the hyperactive mutant HSB17. Genomic DNA was isolated from single neomycin-resistant clones, separated on agarose gels, transferred to nylon, and probed with a radioactive fragment of the neomycin-resistance gene. A 1-kb molecular weight marker is shown on the side of each gel showing DNA in the 3 to 12 kb range.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

8. FIG. 7
Hyperactive transposase improves long-term SEAP activity in mice. C57BL/6–SCID mice were randomly distributed into three groups (n = 4 per group) and received a total of 45 μg of plasmid DNA via tail vein injection. The injected plasmids consisted of a 2:1 molar ratio of transposon (pMSZ-SEAP) to transposase, either a mutant transposase, pTRUF-mHSB (Group 3); the control vector pTRUF-HSB(Group 2); or the hyperactive mutant pTRUF-HSB17 (Group 1). Serum was obtained by tail bleeding at the times indicated (3, 7, 14, 28, 56, and 84 days) and assayed for SEAP activity. The amount of SEAP protein for each sample was quantified by comparison to a standard curve and reported as ng/ml. The data represent the mean SEAP protein levels ± the standard error of measurement.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions