Can the pcDNA5/FRT/TO vector be used for transient overexpression? - (Jul/07/2014 )

Hi,

I have my gene of interest in pcDNA5FRT/TO and would like to generate a stable cell line. Before generating the stable line, however, I would like to test expression by transient transfection.

Can the pcDNA5FRT/TO vector be used for transient transfection? If so, do I need to add doxycycline to induce expression (as would be done with a stable line ...)? In a couple of papers that I found, addition of doxycyclin during transient overexpression of a construct in pcDNA5/FRT/TO was not specifically mentioned.

Thank you,

Norbert

Yes you do need to add dox.  The FRT/TO plasmid has two copies of the tet repressor inserted inside the promoter region, which repress transcription unless Dox is present.

Maybe I am mistaken, but I thought the TO line of pCDNA vectors had two tet operators 3' in the CMV promoter sequence and these repress transcription in a TREX cell line that expresses the Tet Repressor.

Ergo the Tet On system is "On" when Tet binds and inhibits the Tet repressor, or when the Tet repressor is absent, allowing transient overexpression.

I thought the mechanism for these vectors involved de-repressing the expression of your inserted gene in the TREX system, only in the presence of Dox. This enables you to check for expression via transient transfection without any dox dosage optimization. It also means Invitrogen/Life can sell* more Trex cell lines ;)

I am generating my own tet repressor cell line for use with pCDNA4 TO, at least. Maybe frt/TO is different?