Retro-X Tet-One Inducible Expression Systems
Retro-X Tet-One Inducible Expression Systems
Brand: Takara Bio.
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Retro-X Tet-One Inducible Expression Systems
The Retro-X Tet-One Inducible Expression System is a tetracycline-inducible retroviral gene expression system that allows you to produce high titers of recombinant retroviruses for the purpose of establishing a tightly inducible expression system for your gene of interest in a wide variety of dividing mammalian cells. The all-in-one pRetroX-TetOne Vector expresses the Tet-On 3G transactivator from the constitutive human PGK promoter in the forward orientation, and your gene of interest from the PTRE3GS promoter in the reverse orientation. In the presence of doxycycline (Dox), the Tet-On 3G transactivator specifically binds and activates high-level transcription from the inducible promoter that controls expression of your gene. This vector does not contain a selection marker. The kit also includes our Retro-X Universal Packaging System, our premium retroviral packaging system featuring the GP2-293 packaging cell line. All four commonly used envelopes are supplied on separate vectors, including VSV-G, eco, ampho and 10A1, to allow you to choose the tropism that is most appropriate for your target cells.
Overview
- Single-vector format for a simple protocol
- Outstanding induction control and extremely low basal expression for a broad range of dividing cell types
- TRE3G promoter is optimized for very tight transcriptional control
- Supplied with our best-performing retroviral packaging system (Retro-X Universal)
- Two-vector Tet-On 3G format also available
Applications
- Establish a tetracycline-inducible gene expression system in a wide variety of dividing cells using retrovirus
Obtain high fold induction, even from mixed transduced populations
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 hr and assayed for luciferase activity. RLU = relative light units. Clontech always recommends using fetal bovine serum that has been functionally tested because it ensures that you will be able to achieve the full range of inducibility and the lowest background possible with our Tet Systems
Retro-X Tet-One Vector Maps
Retro-X Tet-One Vector Maps. The only difference between the two vectors is the presence of a puromycin resistance cassette in the pRetroX-TetOne-Puro Vector to allow for selection of stable clones using antibiotic selection. pRetro-TetOne does not contain a selection marker gene, and as a result allows for a larger transgene to be cloned (up to 3.1 kb, compared to ~2.1 kb for pRetroX -TetOne-Puro). Transduced clones created using pRetroX-TetOne can instead be isolated by limiting dilution (as described in the user manual).
Tet-One System Mechanism — The Tet-On 3G transactivator protein is expressed constitutively from the human PGK promoter (PPGK) but is unable to bind to the TRE3G promoter (PTRE3G) in the absence of doxycycline (Dox)
Tet-One System Mechanism — The Tet-On 3G transactivator protein is expressed constitutively from the human PGK promoter (PPGK) but is unable to bind to the TRE3G promoter (PTRE3G) in the absence of doxycycline (Dox). When bound by Dox, supplied in the culture medium, the transactivator undergoes a conformational change, binds to PTRE3G and activates transcription of a transgene cloned downstream.
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, 166–76 (2011).
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Obtain high fold induction, even from mixed transduced populations
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 hr and assayed for luciferase activity. RLU = relative light units. Clontech always recommends using fetal bovine serum that has been functionally tested because it ensures that you will be able to achieve the full range of inducibility and the lowest background possible with our Tet Systems
Retro-X Tet-One Vector Maps
Retro-X Tet-One Vector Maps. The only difference between the two vectors is the presence of a puromycin resistance cassette in the pRetroX-TetOne-Puro Vector to allow for selection of stable clones using antibiotic selection. pRetro-TetOne does not contain a selection marker gene, and as a result allows for a larger transgene to be cloned (up to 3.1 kb, compared to ~2.1 kb for pRetroX -TetOne-Puro). Transduced clones created using pRetroX-TetOne can instead be isolated by limiting dilution (as described in the user manual).
Tet-One System Mechanism — The Tet-On 3G transactivator protein is expressed constitutively from the human PGK promoter (PPGK) but is unable to bind to the TRE3G promoter (PTRE3G) in the absence of doxycycline (Dox)
Tet-One System Mechanism — The Tet-On 3G transactivator protein is expressed constitutively from the human PGK promoter (PPGK) but is unable to bind to the TRE3G promoter (PTRE3G) in the absence of doxycycline (Dox). When bound by Dox, supplied in the culture medium, the transactivator undergoes a conformational change, binds to PTRE3G and activates transcription of a transgene cloned downstream.
