I have cloned my gene of interest into pLenti6.3/TO/V5-DEST and would like to use one of your T-REx cell lines as the host for the lentiviral construct. Will that work?
Yes, you can use one of our T-REx cell lines as the host for your pLenti6.3/TO/V5-DEST lentiviral construct. However, please note that while the pLenti6.3/TO/V5-DEST vector is a HiPerform lentiviral vector containing the genetic elements WPRE and cPPT for enhancing viral titer and expression; the T-REx cell lines we offer do not contain these elements. Further, you can use these T-REx cell lines only for transient expression, because the Lenti6.3/TO/V5-DEST lentiviral expression construct, and the Tet repressor plasmid (pcDNA6/TR) that is stably integrated within the T-REx cells, both contain the blasticidin selection marker, making stable cell line development not possible.
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.
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 is the advantage of the Flp-In T-REx system over the T-REx system?
The Flp-In T-REx system combines the targeted integration offered by the Flp-In system with the powerful inducible expression offered by the T-REx system. It allows generation of isogenic, inducible, stable cell lines and permits polyclonal selection of these cell lines. Once the Flp-In T-REx host cell line containing an integrated FRT site has been created, subsequent generation of Flp-In T-REx cell lines expressing the gene(s) of interest is rapid and efficient.
