Switch-On Expression from the Lowest Basal Levels
The GeneSwitch™ System for inducible mammalian expression is ideal for experiments that require the absolute lowest uninduced expression levels. The expression vector pGene/V5-His provides a minimal promoter, GAL4-E1b, consisting of the binding sites for the yeast Gal4 DNA binding protein followed by the TATA sequence from the Adenovirus E1b promoter. Without additional factors, the GAL4-E1b promoter is transcriptionally silent.

To activate transcription from the GAL4 -E1b promoter, the GeneSwitch™ regulatory protein is expressed from a minimal TK promoter on the pSwitch vector. The GeneSwitch™ protein has three functional domains:

• Gal4 DNA Binding Domain (Gal4-DBD) to bind the regulatory protein to the GAL4-E1b promoter
• Truncated human Progesterone Receptor Ligand Binding Domain (hPR-LBD) that undergoes a conformational change when it binds the progesterone antagonist, mifepristone
• Transcription activation domain from the NFØB transcription factor p65 (p65AD) to activate transcription from the silent GAL4-E1b promoter

In the absence of mifepristone, the conformation of the hPR-LBD region prevents the GeneSwitch™ regulatory protein from activating transcription from the GAL4-E1b promoter. When mifepristone is added and binds the hPR-LBD region, the GeneSwitch™ regulatory protein assumes a conformation that permits it to stimulate transcription from the GAL4-E1b promoter (Figures 1 and 2).

Induction of the GeneSwitch™ System leads to activation of the gene of interest on the vector pGene/V5-His. In addition, four Gal4 binding sites upstream of the minimal HSV TK promoter on pSwitch can bind the pSwitch regulatory protein. Therefore, adding mifepristone up-regulates production of the regulatory protein (Figure 1). The increased levels of the GeneSwitch™ regulatory protein result in induction of the gene of interest from pGene/V5-His to levels that can approach those of viral promoters.

The GeneSwitch™ Kit offers exceptionally low uninduced and high induced expression levels in mammalian cells. The system provides the expression vector pGene/V5- His with the minimal, synthetic GAL4-E1b promoter that is transcriptionally silent until activated. The GeneSwitch™ regulatory protein binds the GAL4-E1b promoter to activate transcription upon the addition of mifepristone . In addition, the GeneSwitch™ protein upregulates its own expression, leading to amplified expression of the gene of interest from pGene/V5-His. pGene/V5-His offers several features that facilitate expression analysis and purification of recombinant proteins in mammalian cells:

• C-terminal V5 epitope tag for detection with Anti-V5 Antibodies
• C-terminal polyhistidine (6xHis) tag for rapid purification with nickel-chelating resin and detection with Anti-His(C-term ) Antibodies
• The Zeocin™ resistance gene for effective selection in both mammalian cells and E. coli