Takara Lenti-X Tet-Off Advanced Inducible Expression System \Each

The Lenti-X Tet-Off Advanced Inducible Expression System includes the Lenti-X Tet-Off Advanced Vector Set, Lenti-X Packaging Single Shots (VSV-g), and a sample of Lenti-X GoStix. These materials allow you to produce exceptionally high titers of recombinant, VSV-g pseudotyped lentiviruses for the purpose of establishing an inducible expression system for your gene of interest (GOI) in both dividing and non-dividing cell types. The system provides enough reagents for 16 packaging reactions.

LVX-Tet-Off Advanced (regulator) lentivirus expresses the tetracycline-dependent transcriptional activator, tTA-Advanced. In the absence of doxycycline (Dox), tTA-Advanced specifically activates high-level transcription from thePTightinducible promoter that controls expression of your GOI in your customized LVX-Tight-Puro-GOI (response) lentivirus. In cells cotransduced by both lentiviruses, your GOI will be expressed to high levels in the absence of Dox. The system is maintained in the inactive "off" state in the presence of Dox.

Protocol schematic for creating a tetracycline-inducible lentivirus system.Use the Lenti-X HTX Packaging System and 293T cells to generate high-titer lentiviral supernatants from the pLVX-Tet-On Advanced Vector, and from the pLVX-Tight-Puro Vector containing your gene of interest. Simultaneously coinfect cultures of your target cells with the two lentiviruses (~8 hr). Then, after culturing for an additional 48–72 hr (+/– Dox), harvest the cells for analysis. These same procedures also apply to the Lenti-X Tet-Off Advanced systems, but the effects of Dox are reversed.

Induced expression in the Tet-Off Advanced and Tet-On Advanced systems.The Tet-controlled transactivators are fusion proteins that contain a DNA-binding TetR domain joined to three minimal transcription activation domains from HSV VP16. Each transactivator has been optimized for expression in mammalian cells. In Tet-Off Advanced systems, the basal state is maintained in the presence of doxycycline (Dox), and induced by its withdrawal. Tet-On Advanced systems are activated in the presence of Dox. System induction produces high-level transcription of your gene fromPTight.