Lentiviral Tet-One Inducible Expression Systems
Lentiviral Tet-One Inducible Expression Systems
Brand: Takara Bio.
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Lentiviral Tet-One Inducible Expression Systems
The Lenti-X Tet-One Inducible Expression System is a tetracycline-inducible lentiviral gene expression system that allows you to produce high titers of recombinant, VSV-G-pseudotyped lentiviruses for the purpose of establishing a tightly inducible expression system for your gene of interest in a wide variety of dividing and non-dividing mammalian cells. The all-in-one pLVX-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 Lenti-X Packaging Single Shots (VSV-G), a complete fourth generation lentiviral packaging system, and a sample of Lenti-X GoStix.
Overview
- Highly efficient Lenti-X Packaging Single Shots (VSV-G), (included) generates high titers with a very high safety profile
- Tightly controlled expression of your gene of interest with the TRE3G promoter
- Simple, single-vector design
- Option to use a puromycin resistance cassette to select for stable clones
- Tet-On 3G lentiviral systems are also available
- Tetracycline-inducible expression in the widest range of hard-to-transfect cell types
Seven out of eight Tet-One clones show more than 1,000-fold induction of expression
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 (M.O.I. = 1), and eight individual clones were selected and expanded according to the protocol. Each clone was analyzed for doxycycline-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
The Tet-One system is highly sensitive to low levels of doxycycline (Dox)
The Tet-One system is highly sensitive to low levels of doxycycline (Dox). HEK 293 cells were infected with LVX-TetOne-Luc lentivirus (MOI = 1) and the transduced pool was treated with 10-fold dilutions of doxycycline. 48 hr after treatment, the cells were harvested, lysed, and Western-blotted using anti-luciferase antibody. Luciferase expression reached maximum levels in the presence of only 10 ng/ml Dox. With the greatly increased sensitivity of the Tet-On 3G system, it is more important than ever that the fetal bovine serum (FBS) you use for your studies is guaranteed to be tetracycline-free, we always use Tet System Approved FBS that has been functionally validated.
Lenti-X Tet-One Vector maps
Lenti-X Tet-One Vector maps. The only difference between the two vectors is the presence of a puromycin resistance cassette in the pLVX-TetOne-Puro Vector to allow for selection of stable clones using antibiotic selection. pLVX-TetOne does not contain a selection marker gene, and as a result allows for a larger transgene to be cloned (up to 4 kb, compared to ~3 kb for pLVX-TetOne-Puro). Transduced clones created using pLVX-TetOne can instead be isolated by limiting dilution (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., Schambach, A., Galla, M., Maetzig, T., Baum, C., Loew R. & Schiedlmeier, B. Retroviral and transposon-based Tet-regulated all-in-one vectors with reduced background expression and improved dynamic range. Hum. Gene Ther. 22, 166–176 (2011).
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Seven out of eight Tet-One clones show more than 1,000-fold induction of expression
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 (M.O.I. = 1), and eight individual clones were selected and expanded according to the protocol. Each clone was analyzed for doxycycline-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
The Tet-One system is highly sensitive to low levels of doxycycline (Dox)
The Tet-One system is highly sensitive to low levels of doxycycline (Dox). HEK 293 cells were infected with LVX-TetOne-Luc lentivirus (MOI = 1) and the transduced pool was treated with 10-fold dilutions of doxycycline. 48 hr after treatment, the cells were harvested, lysed, and Western-blotted using anti-luciferase antibody. Luciferase expression reached maximum levels in the presence of only 10 ng/ml Dox. With the greatly increased sensitivity of the Tet-On 3G system, it is more important than ever that the fetal bovine serum (FBS) you use for your studies is guaranteed to be tetracycline-free, we always use Tet System Approved FBS that has been functionally validated.
Lenti-X Tet-One Vector maps
Lenti-X Tet-One Vector maps. The only difference between the two vectors is the presence of a puromycin resistance cassette in the pLVX-TetOne-Puro Vector to allow for selection of stable clones using antibiotic selection. pLVX-TetOne does not contain a selection marker gene, and as a result allows for a larger transgene to be cloned (up to 4 kb, compared to ~3 kb for pLVX-TetOne-Puro). Transduced clones created using pLVX-TetOne can instead be isolated by limiting dilution (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.
