Tet-On 3G Inducible Expression System (with ZsGreen1)

The Tet-On 3G system is a highly sensitive tetracycline inducible expression system

The Tet-On 3G system is a highly sensitive tetracycline inducible expression system. Following cotransient transfection of pCMV-Tet3G and pTRE3G-Luc in HeLa cells, increasing levels of Dox were added and expression of luciferase was measured using an anti-luciferase antibody (Panel A) and a luciferase assay (Panel B). Induced expression was very high even with Dox concentrations as low as 10 ng/ml, and was detectable even at 0.1 ng/ml Dox.

The Tet-On 3G system demonstrates higher sensitivity to doxycycline than Tet-On Advanced system

The Tet-On 3G system demonstrates higher sensitivity to doxycycline than Tet-On Advanced system. Tet-On 3G and Tet-On Advanced genes were integrated at the same locus in a stable HLF33 cell line expressing luciferase from a TRE promoter. For each of these two double-stable cell lines, induced luciferase expression was measured in response to a range of doxycycline (Dox) concentrations. At 5–10 ng/ml Dox, induced expression was 100– to 150-fold higher for the Tet-On 3G cell line, and at 50 ng/ml, expression was 4.6-fold higher (data kindly provided by Professor W. Hillen, and Dr. C. Berens, University of Erlangen)

The PTRE3G promoter results in significantly reduced basal expression

The PTRE3G promoter results in significantly reduced basal expression. HEK 293 cells were transiently cotranstected with both the response vectors (containing luciferase) and regulator vectors from each of the Tet-On 3G and Tet-On advanced inducible expression systems. The cells were cultured in the presence and absence of Dox, and after 24 hr, luciferase expression was measured. Although both systems provided strong expression in the presence of Dox, the Tet-On 3G system produces far lower background expression in the absence of Dox (inset).