Tet-One technology overview

An inducible expression system that's easy to use

Until now, the best inducible mammalian expression systems have all required a two-vector format. Tet-One inducible expression systems are the only commercially available single-vector systems that can deliver 3–4 orders of magnitude difference in expression levels between induced and uninduced states. These systems are available in lentiviral-, retroviral-, and plasmid-based formats.

• No need to create double-stable cell lines
• Obtain >10,000-fold induction
• Viral systems include a high-titer packaging system

The "all-in-one" design of Tet-One systems

Before Tet-One systems were developed, our Tet-On and Tet-Off products all required two separate vectors to introduce the transactivator protein and the inducible promoter controlling your gene of interest, respectively, into your target cells. Tet-One systems provide both of these components on a single vector. The Tet-On 3G transactivator is expressed in the forward direction from the human phosphoglycerate kinase 1 promoter (P_hPGK), and the cloned gene of interest is expressed from the TRE3GS promoter (P_TRE3GS) in the reverse orientation (Figure 1).

Elements of Tet-One systems

The Tet-On 3G transactivator protein

Based on the transcriptional regulators described by Gossen and Bujard, 1992; Gossen et al. 1995; and Urlinger et al. 2000; Tet-On 3G is a modified form of the Tet-Advanced transactivator protein which has been altered to display far higher sensitivity to doxycycline (Dox) (Zhou et al. 2006). Tet-One systems typically reach maximum expression with only 10 ng/ml Dox (Figure 2), approximately two orders of magnitude lower than required for the first generation of Tet-On systems.

P_TRE3GS inducible promoter

The inducible promoter P_TRE3GS provides for very low basal expression and high maximal expression after induction (Loew et al. 2010). It consists of seven repeats of a 19-bp tet operator sequence located upstream of a minimal promoter. P_TRE3GS is a version of the TRE3G promoter (P_TRE3G) that was modified for higher performance in a single vector context. In the presence of Dox, Tet-On 3G binds specifically to P_TRE3GS and activates transcription of the downstream GOI. P_TRE3GS lacks binding sites for endogenous mammalian transcription factors, so it is virtually silent in the absence of induction.

Doxycycline (Dox)

Dox is a synthetic tetracycline derivative that is the effector molecule for all Tet-On and Tet-Off Systems. When bound by Dox, the Tet-On 3G protein undergoes a conformational change that allows it to bind to tet operator sequences located in the P_TRE3GS promoter and activate transcription (Figure 1, bottom panel). The Dox concentrations required for induction are far below cytotoxic levels for either cell culture or transgenic studies, and Tet-On 3G responds to even lower concentrations than its predecessors (Zhou et al. 2006). Note that Tet-On systems (including Tet-One) respond well only to doxycycline, and not to tetracycline (Gossen and Bujard 1992).

Why didn't we recommend previous iterations of all-in-one Tet vectors?

All previously published all-in-one vector designs that we have tested at Takara Bio showed a low signal-to-noise ratio, typically providing only 50- to 100-fold induced expression, even in selected clones. In order to ensure that users could achieve maximum control of gene expression in mammalian cells, we used to recommend the two vector design. Tet-One systems, however, are based on an all-in-one design that has shown up to 25,000-fold induction over background (Heinz et al. 2011). This means that if you use a Tet-One system, there is no need to perform two rounds of clonal screening; you can transfect/transduce and screen a few stable clones and expect to find clones with 10,000-fold induction or more (Figure 3).

Do I always have to screen clones?

No, you don't. If you are working with primary cells, you might not have the ability to do a round of clonal selection. In these cases, it is easy to infect a pool of cells using the Lenti-X or Retro-X Tet-One Systems, and still expect more than a 1000-fold induction of expression across the mixed population. This is also true if you are working with rapidly dividing cell lines (Figure 4); however, over multiple passages, you cannot predict which clone will predominate in the culture. To guarantee consistent, high inducibility in long-term studies, we recommend screening a few clones and picking the best one for expansion. In that respect, screening with Tet-One systems is no different than picking clones for consistent expression when using constitutive expression systems.

References

Gossen, M. et al. Transcriptional activation by tetracyclines in mammalian cells. Science 268, 1766–9 (1995).        

Gossen, M. & Bujard, H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl. Acad. Sci. U. S. A. 89, 5547–51 (1992).

Heinz, N. et al. Retroviral and transposon-based tet-regulated all-in-one vectors with reduced background expression and improved dynamic range. Hum. Gene Ther. 22, 166–176 (2011).          

Loew, R., Heinz, N., Hampf, M., Bujard, H. & Gossen, M. Improved Tet-responsive promoters with minimized background expression. BMC Biotechnol. 10, 81 (2010).   

Urlinger, S. et al. Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity. Proc. Natl. Acad. Sci. U. S. A. 97, 7963–8 (2000).       

Zhou, X., Vink, M., Klaver, B., Berkhout, B. & Das, A. T. Optimization of the Tet-On system for regulated gene expression through viral evolution. Gene Ther. 13, 1382–90 (2006).