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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
  • Tet-One Ordering Information

The “All-in-One” Design of Tet-One Systems

Before the Tet-One Systems were developed, Clontech’s 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. The 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 (PhPGK), and the cloned gene of interest is expressed from the TRE3GS promoter (PTRE3GS) in the reverse orientation (Figure 1).

Tet-One single vector inducible expression schematic

Figure 1. Tet-One design. The Tet-On 3G transactivator protein is expressed constitutively from PhPGK, but is unable to bind to PTRE3GS in the absence of doxycycline (Dox; top panel). When bound by Dox (supplied in the culture medium), the transactivator undergoes a conformational change, binds to PTRE3GS, and activates transcription of a transgene cloned downstream (bottom panel).

Elements of Tet-One Systems

The Tet-On 3G Transactivator Protein

Based on the transcriptional regulators described by Gossen & Bujard (1), Gossen et al. (2), and Urlinger et al. (3), Tet-On 3G is a modified form of the Tet-On Advanced transactivator protein which has been altered to display far higher sensitivity to doxycycline (Dox; 4). 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.

Tet-One Systems exhibit high sensitivity to Dox

Figure 2. The Tet-One system is highly sensitive to low levels of Dox. HEK 293 cells were infected with LVX-TetOne-Luc lentivirus (MOI=1), and the transduced pool of cells was treated with 10-fold dilutions of Dox. 48 hours after treatment, the cells were harvested, lysed, and run on a gel. Levels of luciferase were detected by Western blot using an anti-luciferase antibody. Maximum luciferase expression was achieved with just 10 ng/ml Dox.

PTRE3GS Inducible Promoter

The inducible promoter PTRE3GS provides for very low basal expression and high maximal expression after induction (5). It consists of seven repeats of a 19 bp tet operator sequence located upstream of a minimal promoter. PTRE3GS is a version of the TRE3G promoter (PTRE3G) that was modified for higher performance in a single vector context. In the presence of Dox, Tet-On 3G binds specifically to PTRE3GS and activates transcription of the downstream GOI. PTRE3GS 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 PTRE3GS 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 (4). Note that Tet-On Systems (including Tet-One) respond well only to doxycycline, and not to tetracycline (1).

Why Didn’t Clontech Recommend Previous Iterations of All-in-One Tet Vectors?

All previously published all-in-one vector designs that we have tested at Clontech 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 (6). This means that if you use Tet-One 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).

High Dox-induced expression in nearly every clone

Figure 3. Seven out of eight Tet-One clones show more than 1,000-fold induction of expression. HeLa cells were infected with LVX-TetOne-Puro-Luc lentivirus (MOI = 1), and eight individual clones were selected and expanded according to the protocol. Each clone was analyzed for Dox-induced expression of luciferase. Seven of the eight clones demonstrated more than 1,000-fold induced expression, including two clones with 5,000-fold induction, and one clone with 10,000-fold induction.

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.

Tet-inducible expression using retrovirus

Figure 4. Obtain high fold induction, even from mixed transduced populations. 293T and HepG2 cells were infected with RetroX-TetOne-Luc retrovirus engineered for inducible luciferase expression (MOI = 1). The transduced pools of cells were grown ± Dox for 48 hours and assayed for luciferase activity. RLU = relative light units.

References

  1. Gossen, M. & Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89(12):5547–5551.
  2. Gossen, M. et al. (1995) Science 268(5218):1766–1769.
  3. Urlinger, et al. (2000) Proc. Natl. Acad. Sci. USA 97(14):7963–7968.
  4. Zhou et. al,. (2006) Gene Ther. 13(19):1382–1390.
  5. Loew, R. et al. (2010) BMC Biotechnol. 10:81.
  6. Heinz et al. (2011) Human Gene Ther. 22(2):166–176.

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