1. Volume 2, Issue 3, Pages 276-287 (September 2000)
Ligand-Dependent Regulation of Vascular Endothelial Growth Factor and Erythropoietin Expression by a Plasmid-Based Autoinducible GeneSwitch System 
Ronald V. Abruzzese, Debra Godin, Vidya Mehta, Jerry L. Perrard, Martha French, Wendy Nelson, Gaylen Howell, Michael Coleman, Bert W. O'Malley, Jeffrey L. Nordstrom 
Molecular Therapy 
Volume 2, Issue 3, Pages (September 2000)
DOI: /mthe
Copyright © 2000 American Society for Gene Therapy Terms and Conditions

2. FIG. 1
Structure of GeneSwitch (A) and inducible (B) plasmids. Model of action for the autoinducible GeneSwitch system (C). In the absence of MFP, there is low-level expression of the GeneSwitch protein from the minimal thymidine kinase (tk) promoter. Upon addition of MFP, GeneSwitch protein is activated to induce both itself and the transgene reporter to a high level of expression.
Molecular Therapy 2000 2, DOI: ( /mthe )
Copyright © 2000 American Society for Gene Therapy Terms and Conditions

3. FIG. 2
Induction of secreted alkaline phosphatase in COS-1 (A), HeLa (B), and NIH3T3 (C) cells. Cells were transfected with mixtures of pGS1158 and pAP1205 or pGS1382 and pAP1205. The ratio of GeneSwitch to inducible SEAP plasmid was 1:1 or 10:1. Open bars are SEAP levels obtained in the absence of MFP, while the closed (pGS1158) and lightly shaded (pGS1382) bars indicate the levels of SEAP obtained in the presence of MFP. pAP1166 (CMV-SEAP) (striped bar) was included for each cell line. The number above each pair of bars indicates the fold-induction of SEAP levels. Data are mean values ± SD of three independent transfections.
Molecular Therapy 2000 2, DOI: ( /mthe )
Copyright © 2000 American Society for Gene Therapy Terms and Conditions

4. FIG. 3
Regulation of hVEGF protein in vitro. 293 cells were transfected with a 1:1 mixture of pGS1158/pVF1207 and pGS1382/pVF1207. Twenty-four hours after addition of MFP, cell culture supernatants were taken and assayed for hVEGF protein levels in the absence (open bars) or presence (closed bars) of MFP. Expression from pVF1164 (CMV-hVEGF) (striped bar) and pVF1207 (regulated hVEGF plasmid) are also shown. The number above each pair of bars indicates the fold-induction of hVEGF protein. Data are mean values ± SD of three independent transfections. Asterisks indicate data points for undetectable levels of hVEGF protein.
Molecular Therapy 2000 2, DOI: ( /mthe )
Copyright © 2000 American Society for Gene Therapy Terms and Conditions

5. FIG. 4
In vivo comparison of SEAP regulation by the CMV and autoinducible GeneSwitch systems. pGS1158/pAP1205 or pGS1382/pAP1205 plasmid mixtures were delivered to the hind-limb muscles of C57BL/6 mice on day 0 (n = 10). Five animals from each plasmid group were then implanted subcutaneously with a slow release MFP pellet (open bars) or left untreated (closed bars). Six days later serum samples were taken and assayed for SEAP activity. The number above each pair of bars indicates the fold induction of SEAP levels. Expression from pAP1166 (CMV-SEAP) (striped bar) and pAP1205 (regulated SEAP plasmid) (lightly shaded bar) are also shown. Each data point is the mean value ± SEM of a group of five animals.
Molecular Therapy 2000 2, DOI: ( /mthe )
Copyright © 2000 American Society for Gene Therapy Terms and Conditions

6. FIG. 5
Regulation of hVEGF protein in vivo. pGS1382/pVF1207 plasmid mixtures were formulated with 6 mg/ml sodium poly (L-glutamate), which we have shown to be superior to naked DNA or PVP with electroporation (data not shown), and delivered to the tibialis anterior muscle of male CD-1 mice on day 0 using electroporation. MFP was administered at 0.33 mg/kg on days 5 and 6 (first induction), 11 and 12 (second induction) or 18 and 19 (third induction). The (+) and (–) symbols indicate whether a particular group was treated (+) or not treated (-) with MFP during each induction period. The tibialis muscles were harvested from a group of animals (n = 4) on days 7, 13, and 20 and assayed for hVEGF protein levels. The column labeled Serum shows hVEGF levels obtained from the serum samples of all the animals harvested on day 20 (week 3), untreated and treated with MFP. The severity of hind-limb swelling is noted above each bar, with + being slight, and being massive swelling reaching up into the hindquarters with noticeable interference of hind-limb movement. Each data point is the mean ± SEM of a total of eight tibialis muscles (two/animal). Asterisks indicate data points for undetectable levels of hVEGF protein.
Molecular Therapy 2000 2, DOI: ( /mthe )
Copyright © 2000 American Society for Gene Therapy Terms and Conditions

7. FIG. 6
Regulation of expression of erythropoietin in vivo. A mixture of pGS1382 and pEP1442 were delivered to the hind-limb muscles of C57BL/6 mice on day 0 and MFP was administered orally at 0.33 mg/kg (closed circles) on the indicated days (closed triangles) or left untreated (open circles). Blood hematocrit levels were followed over time. MFP was administered to the untreated animals (open circles) on the indicated days (closed triangles), and hematocrit levels were followed over time in the figure insert. mEpo protein levels were assayed from serum taken on days 13 and 56 (24 h after last MFP administration) and are indicated over the corresponding hematocrit measurement for each treatment. Each data point is the mean ± SEM of a group of four animals. Asterisks indicate data points that are statistically different (Student's t test, P < 0.05).
Molecular Therapy 2000 2, DOI: ( /mthe )
Copyright © 2000 American Society for Gene Therapy Terms and Conditions