The Tet-On Advanced Inducible Gene Expression System is a tightly regulated and highly responsive system that 

produces on-demand, robust expression of your gene of interest （GOI） in target cells. The system is established in 

target cells by sequentially transfecting them with the provided vectors and selecting stable cell lines. Target cells that 

express the Tet-On Advanced transactivator, and that also contain an integrated TRE-based expression vector （e.g., 

pTRE-Tight） will express high levels of your GOI when cultured in the presence of the system’s inducer, doxycycline 

（Dox） （Figure 1）.

B. Elements of Tet-On Advanced Induction

Based on the original tetracycline （Tc）-regulated transcriptional transactivators described by Gossen & Bujard 

（1992） and Gossen et al. （1995）, Tet-On Advanced is a modified transactivator protein that is optimized for expression in mammalian cells, and which demonstrates higher sensitivity and fidelity than previous versions （Urlinger, et 

al. 2000）. The inducible promoter, P

Tight, provides for very low basal expression and tightly controlled induction. 

• The Tet-On Advanced transactivator. The pTet-On Advanced vector constitutively expresses the tetracyclinecontrolled transcriptional transactivator, Tet-On Advanced （Urlinger et al., 2000）. This engineered protein 

consists of a mutant E. coli TetR protein （rTetR） fused to three minimal “F”-type activation domains derived 

from the herpes simplex virus VP16 protein （Baron et al., 1997, Triezenberg et al., 1988）. In the presence 

of Dox, Tet-On Advanced binds to the tetO sequences in P

Tight, and activates high level transcription from 

this inducible promoter. The Tet-On Advanced coding sequence is fully synthetic and utilizes human codon 

preferences to increase its expression level and stability in mammalian cells. 

• The P

Tight inducible promoter. This is an inducible promoter that controls transcription of your GOI. The 

P

Tight composite promoter was originally developed as the Ptet-14 promoter in the laboratory of Dr. H. Bujard 

and consists of a modified Tet-Responsive Element （TREmod） containing 7 direct repeats of the tet operator 

sequence, tetO, which is joined to a minimal CMV promoter （P

minCMV∆）. P

Tight lacks binding sites for endogenous mammalian transcription factors, so it is virtually silent in the absence of induction. In the presence of 

Dox, Tet-On Advanced binds tightly and specifically to P

Tight and activates transcription of the downstream 

GOI （Figure 1）.