I used one of your T-REx cell lines and am getting expression of my gene of interest in the absence of inducer. Is there any workaround for this problem?
Almost all lots of FBS contain tetracycline, because FBS is generally obtained from cows that have been fed a diet containing tetracycline. If cells are cultured in medium containing FBS that is not reduced in tetracycline, there may be low basal expression of the gene of interest in the absence of added tetracycline. In such cases, we recommend purchasing tetracycline-reduced FBS from our Gibco Cell Culture Division. To be qualified as tetracycline-reduced, these lots must contain below 19.7 ng/mL tetracycline (which happens to be the assay detection limit).
Note: The binding constant for Tet-repressor protein with tetracycline is 3 nM. Assuming that the medium contains 10% serum, a serum tetracycline concentration of 19.7 ng/mL is equivalent to 4 nM tetracycline. Thus, keep in mind that it is possible to get basal level expression even from tetracycline-reduced FBS.
Is multiple integration of the Flp-In expression construct possible? How do you screen for multiple integrants, and how stable is the Flp-In expression cell line?
In theory, one can get multiple integrations of the Flp-In expression construct—an FRT-specific integration event and a random, second-site integration. However, random integration is a relatively uncommon event. Limiting the amount of DNA in the transfection will reduce the chance of second-site integration. We have transfected 293 cells (lacking the FRT site) with the pcDNA5/FRT vector and have identified one potential second-site integrant after screening over 200 clones. DNA integrations can be detected by Southern blot. A single integrant will display a single band; double: two; triple: three, etc. We have maintained a number of Flp-In expression cell lines for over four months and have not observed any loss of the Flp-In expression construct, whether hygromycin selection was maintained or not.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Why is sequential transfection recommended over co-transfection in the T-REx and GeneSwitch systems?
When a co-transfection is performed, there is no way of testing the double stable cell line for functional TetR or GeneSwitch protein, respectively. On the other hand, when sequential transfection is performed, one can functionally test the generated T-REx or GeneSwitch cell line by transiently transfecting the lacZ expression control plasmid and then picking a clone that shows the lowest basal level of expression of lacZ in the absence of the inducer, and the highest level of lacZ in the presence of the inducer. This clone can then be expanded and used to transfect the T-REx or GeneSwitch expression construct, as the case may be.
What is the main advantage of the GeneSwitch system over the T-REx system? And what is its main disadvantage?
With the GeneSwitch system, it is possible to have the absolute lowest basal levels of expression of the gene of interest, whereas the T-REx system may be a little leaky due to the inevitable presence of tetracycline in FBS. The induced level of expression in the GeneSwitch system can be even higher than that seen with the CMV promoter. The disadvantage of the GeneSwitch system is that the expression does not appear to switch off very easily in culture, although it has been demonstrated to function beautifully in transgenics. The T-REx system, on the other hand, can be switched on and off by the addition and removal of the inducer.
What kind of Flp-In T-REx cell lines do you offer?
We offer the Flp-In T-REx system that contains pFRT/lacZeo and pcDNA6/TR stably integrated into HEK 293 cells. This cell line has been functionally tested for its ability to regulate expression.
