Are you comparing to non induced or to non infected cells? Some batches of serum have tet in them and can result in relatively high basal activity and as a result low fold of induction. In addition, do you add dox throughout your 5 day experiment? Dox is supposedly stable in the medium for ≈48hrs so if you just add once by day 5 your cells are without dox for 3 days and you may be losing your induction due to stopped expression and decay of you product, Dox induction is usually very fast so there is no reason you won't be able to test induction a lot sooner. One last thing, looking up the FUW system it seem you need to co-infect with viruses expressing the tet transactivator, did you prep these at the same time and do you have an indication as to the level of the tetTR expression?

I typically start off with lower levels of Dox and work my way up to the max dose. You don't kill so many cells that way to begin with.

Are you comparing to non induced or to non infected cells? Some batches of serum have tet in them and can result in relatively high basal activity and as a result low fold of induction. In addition, do you add dox throughout your 5 day experiment? Dox is supposedly stable in the medium for ≈48hrs so if you just add once by day 5 your cells are without dox for 3 days and you may be losing your induction due to stopped expression and decay of you product, Dox induction is usually very fast so there is no reason you won't be able to test induction a lot sooner. One last thing, looking up the FUW system it seem you need to co-infect with viruses expressing the tet transactivator, did you prep these at the same time and do you have an indication as to the level of the tetTR expression?

Thanks a lot for the replies!

I have compared it with induced and non-induced cells.

I am going to redo my experiment by testing the induction sooner. Good to hear that the system can give up to 150-fold induction.

One of the issues you should take into account, that unfortunately happens from time to time is that the serum in the media contains tetracycline (the guys preparing the FCS sometimes they treat the cows with tetracycline before getting the fetus). This gives a partial induction on the untreated cells, which although not optimal, does not allow a large (60-fold with our plasmids) induction when you add the tracycline or the dox.

I would suggest you to check the sera, or simply change the sera or buy sera that is sold as tetracycline free

I agree with Jose that FCS tetracycline may be the problem and you can use Tet-free FCS or KOSR (if applicable for your cells).

As Yoav said you need to coexpress a rtTA along with your transgene under FUW vector. This may lead to some considerations:

• The induction rate (depends on cell type and virus titer) of both rtTA and transgene lentiviruses should be high enough to support duble-transgenesis of the cells. You also may use double antibiotic selection to isolate double-infected cells.
• rtTA expression may be low due to cell type dependent weak promoter activity (I had such an experience with low CMV-derived rtTA expression in embryonic stem cells).
• There is also a possibility of epigenetic inactivation of viral transgenes in certain cell types. This may be more evident at higher passage numbers and I believe to be the hardest obstacle to deal with!

However, I don't want to freak you out, because these problems are not so evident and TET-ON systems are usually easy to setup and use.

Thanks a lot! I am going to use Tet-free FBS for my subsequent experiments!