1. Endothelial targeting of the Sleeping Beauty transposon within lung
Li Liu, Sonia Sanz, Arnold D. Heggestad, Vijay Antharam, Lucia Notterpek, Bradley S. Fletcher 
Molecular Therapy 
Volume 10, Issue 1, Pages (July 2004)
DOI: /j.ymthe
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

2. Fig. 1
Enhanced transposition of the ET-1-driven transposons. (A) Schematic representation of the various ET-1 transposon constructs. Inverted repeat/direct repeat elements (IR/DR) flank the neomycin expression cassettes driven by the indicated promoters. The right-side IR/DR element has been modified (gray) in plasmids pEM, pMSF, and pMSZ. Endo, ET-1; pA, poly(A) sequence. (B) Transposition assays of the transposon constructs in HeLa cells. The y axis is fold transposition relative to the cotransfection of the constructs pT-SVNeo and pCMV-SB. In these studies, equal molar ratios of transposon/transposase were transfected (0.46 nmol) to allow comparisons between the constructs. (C) Transposition assay within HUVE cells. The number of actual neomycin-resistant colonies is reported on the y axis. The data plotted for B and C represent the means and standard deviations of three independent experiments performed in duplicate.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

3. Fig. 2
Evaluation of ET-1 promoter specificity in various cell lines. Plasmids expressing GFP driven by either the CMV or the ET-1 promoter (Endo) were transiently transfected into the indicated cell types. FACS analysis was performed at 48 h posttransfection and the percentage of GFP-positive cells (% in right upper quadrant), as well as the mean fluorescence intensity (MFI) of the GFP-positive populations, is reported.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

4. Fig. 3
Long-term SEAP activity in mice following delivery of SB transposons. C57Bl/6–SCID mice were randomly classified into four groups (n = 5 per group) and received 30 μg of pMSZ-SEAP transposon with various amounts of pTRUF-HSB, 0 (Group 1), 30 (Group 2), 15 (Group 3), and 2 μg (Group 4), via tail vein injection. Plasmid pcDNA3.1 was used as nonspecific filler to maintain the total DNA at 60 μg. Serum was obtained by tail bleeding at the times indicated (days +1, 2, 3, 5, 7, and 14 and every 2 weeks thereafter up to day +70) 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 © 2004 The American Society of Gene Therapy Terms and Conditions

5. Fig. 4
GFP expression and colocalization studies in the mouse lung following SB-mediated gene delivery. Animals received 60 μg of plasmid DNA (40 μg of pMSZ-GFP and 20 μg of pTRUF-HSB) via tail vein injection and were sacrificed at 3 days or 6 weeks postinjection. Lungs were fixed and sectioned as described under Materials and Methods. Sections from (A) control animals and (B) 3 days or (C) 6 weeks postinjection were evaluated for GFP expression. Parallel sections were stained with von Willebrand factor (vWF) (a marker of endothelial cells) or surfactant protein A (SP-A) (a marker of type II pneumocytes) antibodies. The GFP and (D, F) anti-vWF or (E, G) anti-SP-A images were merged to visualize cells expressing both molecules. The nuclei were counterstained with Hoechst dye for total cell number quantification. All images were at 60× original magnification. (H) Percentage of GFP-positive cells was quantified at both the 3-day and the 6-week time points and is expressed as percentage of GFP-positive cells ± SD. (I) Percentage of transgene expression colocalizing with either endothelial cells or type II pneumocytes. The data are expressed as percentage of GFP-positive cells ± SD.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions