Frequently Asked Questions
Since this gene regulation system consists of two plasmids, what is the ratio used in vitro and in vivo?
What cell lines has the system been tested in?
The GeneSwitch and inducible transgene plasmids are used in a one-to-one ratio.
What concentration of Mifepristone (MFP) is used in vitro?
We have tested the system in HeLa, COS-1, NIH3T3, A549, HepG2, C2C12 and CHO cells. The only cell line where the system doesn't seem to function well is CHO (induction of the reporter is usually less than two-fold).
Can the GeneSwitch gene and inducible transgene be placed on the same plasmid?
We routinely use 10-8 M MFP with cultured cells.
Does the method of transfection affect the performance of the GeneSwitch® system?
We have made limited attempts to put both components of the system on the same plasmids. All of the resulting plasmids showed an increase in basal (absence of MFP) reporter activity, which decreased the fold induction levels.
Does the amount of plasmid DNA used
in the transfection effect the performance of the GeneSwitch® system?
We have found that the method of transfection does affect the system. We recommend using a non-lipid transfection reagent, such as CaCl2,
DEAE-dextran or FuGENE 6.
Where can I get the GeneSwitch® system?
We have found that overexpression of the GeneSwitch protein increases basal expression, which lowers fold induction levels. Therefore, we recommend using no more than 300 ng of each plasmid when transfecting 1-5 x 105 cells.
Is there a commercial source for Mifepristone (MFP)?
It is commercially available through Invitrogen.
Are the plasmids made available through Invitrogen identical to the system described on the website?
Yes, it is available from Sigma-Aldrich, Co.
No, the plasmids made available through Invitrogen have a different backbone than the ones used at Inovio. pSwitch and pGene/V5-His (Invitrogen plasmids) contain additional eukaryotic transcription units driven by the SV40 promoter (Hygromycin resistance for pSwitch and Zeocine resistance for pGene/V5-His) for effective selection in mammalian cells. Both Invitrogen plasmids contain the Ampicillin resistance gene for bacterial selection. The pGene/V5-His plasmid also contains a C-terminal polyhistidine (6xHis) tag for rapid purification or detection (with Anti-His (C-term) antibodies) of the induced transgene of interest. The Inovio plasmid backbone is based on pUC-18 with the Ampicillin resistance gene replaced by the Kanamycin resistance gene.