Tet on 3g tetracycline inducible gene expression system

The Tet-On 3G tetracycline inducible gene expression system (Clontech, Ozyme, Saint Quentin en Yulines, France) was used for generation of stable doxycycline inducible clones of MTG16 inserted under the TRE3G promoter (P_TRE3G) in B-lymphoblastic Raji cells (Raji/MTG16 Tet-On 3G cells) as previously described [1 (link)]. Incubation with 10–20 ng/ml of the tetracycline analog doxycycline induces Tet-On 3 G trans activator binding to tet operator repeats within P_TRE3G leading to transcriptional MTG16 activation. MTG16 biosynthesis was seen after 3 to 4 h of induction at a very low concentration (20 ng/ml) of doxycycline making unspecific effects unlikely (data not shown).

The Tet-On 3G Tetracycline Inducible Gene Expression System (Clontech Laboratories, Inc.) was utilized to generate stable cell lines inducibly expressing HA-α_2B-AR in HEK293 cells as described previously^{17 (link),35} . Briefly, HA-α_2B-AR was cloned into the pTRE3G-TRES vector at the BglII and ClaI restriction sites and co-transfected with the PLKO.1 vector in HEK293 cells using Lipofectamine 2000. Inducible α_2B-AR expression was verified by intact cell ligand binding assays, immunoblotting and confocal microscopy³⁵ . The current study uses a cell line that expresses 8.5 × 10⁵ receptors per cell.

The Tet-On 3 G Tetracycline Inducible Gene Expression System (Clontech Laboratories, Inc.) was utilized to generate stable cell lines inducibly expressing HA-α_2B-AR in HEK293 cells as described previously47 (link). Intact cell ligand binding assays, immunoblotting and confocal microscopy were used to characterize inducible expression of α_2B-AR at the cell surface47 (link). A cell line expressing 8.5 × 10⁵ α_2B-AR per cell after incubation with doxycycline at a concentration of 40 ng/ml for 24 h was utilized in the current study.