The Adeno-X Adenoviral System 3 (Tet-On 3G Inducible) from Takara Bio combines the tightest and most sensitive control of gene expression with the most advanced commercially available adenoviral vector system. With this system, tightly-controlled inducible expression is as easy as constitutive expression, and cloning into an adenoviral vector is as straightforward as cloning into any plasmid.

How does the Tet-On 3G inducible system work?

Target cells that express the Tet-On 3G transactivator protein and contain a gene of interest (GOI) under the control of a TRE3G promoter (P_TRE3G) will express high levels of your GOI, but only when cultured in the presence of doxycycline (Dox), a tetracycline analog. When bound by Dox, the Tet-On 3G protein undergoes a conformational change that allows it to bind to tet operator (tetO) sequences located in P_TRE3G (Figure 1). In contrast to TetR-based systems, Tet-On technologies activate rather than repress transcription, a critical difference which results in far lower basal expression, higher maximal expression, a more rapid response time—and ultimately, makes them the first choice for conditional expression.

What makes this system so easy to use?

1. Simple & efficient cloning—Each kit is supplied with a prelinearized adenoviral plasmid (pAdenoX-Tet3G) and a complete In–Fusion HD Cloning Kit. Simply amplify your gene of interest using primers that contain 15 bp of homology to the vector insertion site and fuse the two linear DNA molecules using In-Fusion (Figure 2). This system makes cloning into the 34-kb pAdenoX-Tet3G vector as simple as cloning into any plasmid.
2. All-in-one vector—The Tet-On 3G transactivator gene has been pre-cloned into the E3 region of the adenoviral genome and is expressed constitutively from a CMV promoter. Clone your gene of interest using In-Fusion HD at the E1 region of the adenovirus between the tightly regulated P_TRE3G promoter and an SV40 polyA signal. Because the two regions are widely separated, interference from the CMV promoter cannot affect basal expression from P_TRE3G and so a very low basal expression and high fold inducibility are retained (Figures 3–5).

Lowest-ever background, highest sensitivity

The combination of two optimized elements makes Tet-On 3G the highest performing inducible expression system.

1. P_TRE3G promoter—mutations have reduced background expression from the inducible promoter to very low levels compared to previous generations of the Tet-On System (Loew et al. 2010).
2. Tet-On 3G transactivator protein—compared to early generations, mutations have significantly increased its sensitivity to the inducer doxycycline (Zhou et al. 2006).

When the two elements are combined, not only can you detect high expression of your protein after exposure to Dox, but you can control the level of expression by titration of the Dox concentration (Figure 3) and you can generate very high fold induction, up to a 3,000-fold difference between the induced and uninduced states (Figure 4). The maximum expression level can be manipulated by increasing the amount of virus per cell (Figure 5).

Figure 5. Expression increases with higher MOIs. HeLa cells were infected with varying MOIs of pAdenoX Tet-On 3G adenovirus that expresses luciferase. After four hours, the media was replaced with fresh media +/- doxycycline (1 μg/ml). Cultures were harvested and assayed for luciferase activity. Maximal expression increases with increasing MOI, which also results in a slight increase in background expression.

Unlike the leading competitor, the Adeno-X system really is easy

Compared to the leading competitor system, which requires eight days or more for a cloning procedure that involves cloning into a shuttle vector and transformation of two different E. coli strains, the Adeno-X system really is easy and allows you to finish cloning with high efficiency in just two to three days. The Adeno-X system does not use a shuttle vector, so no subcloning is needed. A high-performance E. coli strain (Stellar Competent Cells) is included with the kit (see Table I).

Supreme flexibility—create mutations or fusion proteins in a single reaction

The power of In-Fusion Cloning technology enables you to directly join the pAdenoX-Tet3G vector to one or more PCR fragments in a single reaction. This means that in a single cloning reaction you can, for example, fuse your gene of interest to a fluorescent protein/tag or create a point mutation within your gene of interest (Zhu et al. 2007). To do so, you simply amplify 2 PCR products each with 15 bp homology to the pAdenoX- Tet3G vector at one end and 15 bp homology to each other at the other end. The central overlapping region will be at the junction of the fusion protein or at the region of the point mutation (Figure 6).

Figure 6. Create a point mutation and clone in one step using In-Fusion Cloning.

The Adeno-X Adenoviral System 3 (Tet-On 3G Inducible) provides tightly-controlled gene expression in an easy-to-use tetracycline-inducible system. An all-in one adenoviral vector constitutively expresses the Tet-On 3G transactivator and allows simple, efficient In-Fusion HD Cloning of a gene of interest under the control of a P_TRE3G promoter. This optimized transactivator/promoter combination provides exceptionally low background and high sensitivity, resulting in a highly inducible system that generates very high fold-induction and maximal expression levels that increase with increasing MOI. The Adeno-X Adenoviral System 3 (Tet-On 3G Inducible) is easier to use than a leading competitor system because it does not use a shuttle vector and thus requires no subcloning. This system is also quite flexible, allowing the creation of mutations or fusion proteins in a single reaction. 

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

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).            

Zhu, B., Cai, G., Hall, E. O. & Freeman, G. J. In-fusion assembly: seamless engineering of multidomain fusion proteins, modular vectors, and mutations. Biotechniques 43, 354–9 (2007).