1. Volume 15, Issue 7, Pages 1340-1347 (July 2007)
Development and Validation of a Robust and Versatile One-plasmid Regulated Gene Expression System 
Paul Szymanski, Peter J Kretschmer, Maxine Bauzon, Fang Jin, Hu Sheng Qian, Gabor M Rubanyi, Richard N Harkins, Terry W Hermiston 
Molecular Therapy 
Volume 15, Issue 7, Pages (July 2007)
DOI: /sj.mt
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
The two-plasmid GeneSwitch (GS) system.6 Plasmid 1 expresses a transcriptional activator (the GS protein) whose activity is regulated by the small-molecule drug, mifepristone (MFP). Plasmid 2 expresses a target gene in response to the activated GS protein. GAL4 DBD, DNA binding domain of the yeast GAL4 protein; PR LBD, mutated ligand binding domain of the human progesterone receptor; p65 AD, transcriptional activation domain of the human p65 subunit of NF-κB.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
The four pBRES plasmid vectors. The orientations of the two transcription cassettes of pGT1, 2, 3, and 4 are diagrammed. The bacterial plasmid backbone, shown below pGT1 only, has the same orientation, kanamycin resistance gene (KanR) on the left, and the pUC origin of replication (ori) on the right, in all four plasmids. The four pBRES units, transactivator and target gene cassettes together, can be transferred intact to a vector of choice by digestion with flanking restriction enzymes as shown. Restriction sites illustrate the modular nature of the of the pBRES molecules, enabling replacement of component regulatory elements and coding regions. Abbreviations are as follows: 6xGAL4, six copies of 17-base pair GAL4 binding site; MCS, multi-cloning site; hGH pA, human growth hormone polyadenylation element; actin pro, chicken skeletal muscle α-actin promoter; TA, transactivator coding region; SV40 pA, simian virus 40 late polyadenylation element. See Materials and Methods for details of plasmid constructions.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
Activity of pBRES-human interferon-β (pBRES-hIFN) plasmids in vitro and in vivo. (a) In vitro. Mouse C2C12 myoblasts were transfected with a two-plasmid inducible hIFN expression system (pGS pGER129), pBRES-hIFN plasmids pGT27–30, or a cytomegalovirus promoter hIFN expression plasmid (pGER125). Twenty-four hours after transfection the media was replaced with fresh media with/without mifepristone (MFP), and the media was collected 24 hours later for hIFN enzyme-linked immunosorbent assay (ELISA). The average of two independent transfections is shown. Error bars represent the mean of the two transfections + SD. Numbers above + MFP bars indicate fold induction over − MFP. The orientation of the two cassettes of pGT27-30 is indicated below each plasmid name. (b) In vivo. Plasmids were injected with electroporation into mouse tibialis and gastrocnemius muscles. MFP was administered intraperitoneally on days 8–11, and mice were bled on day 11, 6 hours after MFP injection. The serum was assayed for hIFN by ELISA. The results are shown as the mean of five animals per group + SD. pGT74 was not tested in the presence of MFP.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
Multiple cycles of human interferon-β (hIFN) expression from the adeno-associated virus-1 pBRES vector in mice. C57Bl/6 mice were injected in the tibialis and gastrocnemius muscles with a total of 5.0 × 1010 viral particles/mouse on day 0, and treated with mifepristone (MFP) for 4 days in each cycle. Bleeds were taken 3 days before MFP administration, after 4 days of MFP treatment, and again 1 week later for each cycle of induction. The serum was assayed for hIFN by enzyme linked immunosorbent assay. The results are shown as the mean (n = 5) ± SD.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

6. Figure 5
pBRES-human interferon-β (pBRES-hIFN) plasmids with alternative promoters, 5′ untranslated regions (UTRs), and introns. (a) Plasmid structure. UT12: 5′ UTR of the cytomegalovirus immediate-early (CMV IE) gene. IVS8, Synthetic intervening sequence; chEF-1α, Chimpanzee EF-1α promoter, 5′ UTR, and intron; hUbiB, human ubiquitin B promoter. (b) In vitro activity. Mouse C2C12 myoblasts were transfected with the pBRES-hIFN plasmids pGT28, 110, 92, or 94 and 2 days later were differentiated into myotubes for 6 days. Cells were treated with mifepristone (MFP) for 24 hours prior to collection of media for hIFN enzyme-linked immunosorbent assay. Values are expressed as % of pGT28 in the presence of MFP. The average of three independent transfections is shown. Error bars represent the mean of the three transfections + SD.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions