Clontech’s Adeno-X Adenoviral System 3 (Tet-On 3G Inducible) 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 (PTRE3G) 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 PTRE3G (see Images & Data tab). 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, the first choice for conditional expression.
What Makes This System So Easy To Use?
- 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 PTRE3G promoter and an SV40 polyA signal. Because the two regions are widely separated, interference from the CMV promoter cannot affect basal expression from PTRE3G and so a very low basal expression and high fold-inducibility are retained (see Images & Data tab).
- Easy cloning—Until now the main drawback of commercially supplied adenoviral vector systems has been the need to use complex cloning procedures to overcome the difficulties with cloning into large (~34 kb) plasmids. At Clontech, our Adeno-X virologists thought “wouldn’t it be great if you could clone directly into the adenoviral plasmid just like any plasmid?” They then harnessed the power of In-Fusion HD cloning technology to make this happen.
Lowest-Ever Background, Highest Sensitivity
The combination of two optimized elements makes Tet-On 3G the highest performing inducible expression system.
- PTRE3G promoter—mutations have reduced background expression from the inducible promoter to very low levels compared to previous generations of the Tet-On System.
- Tet-On 3G transactivator protein—compared to early generations mutations have significantly increased its sensitivity to the inducer doxycycline. When the two elements are combined, not only can you detect high expression of your protein after exposure to Dox, you can control the level of expression by titration of the Dox concentration and you can generate very high fold induction, up to 3000-fold difference between the induced and uninduced states. Maximum expression level can be manipulated by increasing the amount of virus per cell (see Images & Data tab).
Unlike the Leading Competitor, Adeno-X Really Is Easy
Compared to the leading competitor system, which requires 8 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 2–3 days. Adeno-X uses no shuttle vector so requires no subcloning, and a single high-performance E. coli strain (Stellar) is included with the kit.