Mammalian Selection：Hygromycin

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:

1. Gal4 DNA Binding Domain （Gal4-DBD） to bind the regulatory protein to the GAL4-E1b promoter
2. Truncated human Progesterone Receptor Ligand Binding Domain （hPR-LBD） that undergoes a conformational change when it binds the progesterone antagonist, mifepristone
3. Transcription activation domain from the NF?B transcription factor p65 （p6D） 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.

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. 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:

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