T-REx™ System From Invitrogen

Invitrogen’s T-REx™ System is a tetracycline regulated mammalian expression system. The system utilizes a repressor mechanism to block transcription from the CMV promoter in the absence of tetracycline. The T-REx™ system consists of two components: The pcDNA™6/TR regulatory vector provides high-level expression of the tetracycline repressor (TR) protein and the expression vector which contains the gene of interest (GOI) with the CMV promoter upstream. Inserted between the TATA box of the CMV promoter and the transcriptional start site of your GOI are two tetracycline operator sequences (TetO₂). When mammalian cells are transfected with these two components, expression of TR from the regulatory vector binds to the TetO₂ sequence and represses transcription of your GOI. Addition of tetracycline to the cell culture medium binds with high affinity to and alters the confirmation of TR which is then released from the TetO₂ sites, allowing transcription of the GOI.

The expression vector is available in a number of different formats, standard vectors with multiple cloning sites and Gateway® vectors. Vectors are available which allow for expression of native protein and also with various tags (e.g. C-myc and 6xHis). The regulatory vector contains blasticidin resistance genes to enable selection of stably transfected cells whereas the expression vectors are available with resistance genes for either geneticin (Gateway® vectors), hygromycin or zeocin, hence allowing the generation of cell lines stably transfected with both regulatory and expression vectors. Further convenience is available as Invitrogen also sell a number of cell lines (HEK-293, CHO, HeLa and Jurkat) already stably transfected with pcDNA™6/TR regulatory vector.

Utilization of the T-REx™ System begins with the subcloning of the GOI into one of the inducible expression vectors. This construct is then co-transfected with the regulatory plasmid into the mammalian cell of choice. 48 hrs after transfection, cells are treated with tetracycline, causing the TR to detach from the hybrid CMV/ TetO₂ promotor in the inducible expression vector which allows transcription of the GOI to occur. Stable cell lines expressing the Tet repressor from pcDNA™6/TR can be established (or purchased from Invitrogen) for use with different GOI/inducible expression vector constructs.

We have had great success using the T-REx™ System to express proteins which we found to be toxic or inhibitory to cells when overexpressed in traditional expression systems. In most cases, we have used T-REx™ cell lines generated by Invitrogen to stably express the Tet repressor and transfected those with our GOI inserted in a T-REx™ inducible expression vector (we have used almost exclusively the Gateway® version of these vectors). We use Lipofectamine 2000 to transfect the cells and have always obtained a high transfection efficiency. We generally establish stable cell lines and use the resistance gene for geneticin for this purpose. We have observed very low leaky expression of the GOI in the absence of tetracycline but obtain high levels of expression at 24-48 hrs after addition of tetracycline to the cell culture medium.