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 P_TRE3GS 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.

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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

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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.

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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).

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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 (P_PGK) but is unable to bind to the TRE3G promoter (P_TRE3G) in the absence of doxycycline (Dox). When bound by Dox, supplied in the culture medium, the transactivator undergoes a conformational change, binds to P_TRE3G and activates transcription of a transgene cloned downstream.

Required Products

Cat. #	Product	Size		License	Quantity	Details
631106	Tet System Approved FBS	500 mL			*
Fetal Bovine Serum (FBS) that has been functionally tested for optimal use with all of our Tet Systems. This FBS does not contain trace levels of tetracycline (or its derivatives) which have been observed to interfere with proper regulation of TRE-controlled gene expression. Use of this serum ensures the maximal range of induction possible with the Tet Systems. Documents Components Image Data Back Fetal bovine serum (FBS) approved for use with tetracycline-inducible expression systems Fetal bovine serum (FBS) approved for use with tetracycline inducible expression systems. Only Takara Bio USA functionally tests FBS to be sure that no contaminating tetracycline derivatives interfere with induced expression levels. Back Our fetal bovine serum is functionally tested to guarantee consistent results with tetracycline-inducible expression systems Fetal bovine serum from Clontech is functionally tested to guarantee consistent results with tetracycline-inducible expression systems. Serum from other vendors that have not been functionally tested may contain tetracyclines that affect the maximum expression in Tet-Off systems, or generate background in Tet-On systems. Data here shows induction of luciferase expression from our CHO-AA8-Luc Tet-Off Control Cell Line using different sources of FBS.
631101	Tet System Approved FBS, US-Sourced	500 mL			*
Fetal Bovine Serum (FBS) that has been functionally tested for optimal use with all of our Tet Systems. This FBS does not contain trace levels of tetracycline (or derivatives) which have been observed to interfere with proper regulation of TRE-controlled gene expression. Use of this serum ensures the maximal range of induction possible with the Tet Systems. This FBS has been processed in the United States. Documents Components Image Data Back Fetal bovine serum (FBS) approved for use with tetracycline-inducible expression systems Fetal bovine serum (FBS) approved for use with tetracycline inducible expression systems. Only Takara Bio USA functionally tests FBS to be sure that no contaminating tetracycline derivatives interfere with induced expression levels. Back Our fetal bovine serum is functionally tested to guarantee consistent results with tetracycline-inducible expression systems Fetal bovine serum from Clontech is functionally tested to guarantee consistent results with tetracycline-inducible expression systems. Serum from other vendors that have not been functionally tested may contain tetracyclines that affect the maximum expression in Tet-Off systems, or generate background in Tet-On systems. Data here shows induction of luciferase expression from our CHO-AA8-Luc Tet-Off Control Cell Line using different sources of FBS.

The Lenti-X Tet-One Inducible Expression System (Puro) 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-Puro Vector expresses the Tet-On 3G transactivator from the constitutive human PGK promoter in the forward orientation and your gene of interest from the P_TRE3GS 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. In addition, this vector contains a puromycin antibiotic 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.

Back

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

Back

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.

Back

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).

Back

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 (P_PGK) but is unable to bind to the TRE3G promoter (P_TRE3G) in the absence of doxycycline (Dox). When bound by Dox, supplied in the culture medium, the transactivator undergoes a conformational change, binds to P_TRE3G and activates transcription of a transgene cloned downstream.

Required Products

Cat. #	Product	Size		License	Quantity	Details
631106	Tet System Approved FBS	500 mL			*
Fetal Bovine Serum (FBS) that has been functionally tested for optimal use with all of our Tet Systems. This FBS does not contain trace levels of tetracycline (or its derivatives) which have been observed to interfere with proper regulation of TRE-controlled gene expression. Use of this serum ensures the maximal range of induction possible with the Tet Systems. Documents Components Image Data Back Fetal bovine serum (FBS) approved for use with tetracycline-inducible expression systems Fetal bovine serum (FBS) approved for use with tetracycline inducible expression systems. Only Takara Bio USA functionally tests FBS to be sure that no contaminating tetracycline derivatives interfere with induced expression levels. Back Our fetal bovine serum is functionally tested to guarantee consistent results with tetracycline-inducible expression systems Fetal bovine serum from Clontech is functionally tested to guarantee consistent results with tetracycline-inducible expression systems. Serum from other vendors that have not been functionally tested may contain tetracyclines that affect the maximum expression in Tet-Off systems, or generate background in Tet-On systems. Data here shows induction of luciferase expression from our CHO-AA8-Luc Tet-Off Control Cell Line using different sources of FBS.
631101	Tet System Approved FBS, US-Sourced	500 mL			*
Fetal Bovine Serum (FBS) that has been functionally tested for optimal use with all of our Tet Systems. This FBS does not contain trace levels of tetracycline (or derivatives) which have been observed to interfere with proper regulation of TRE-controlled gene expression. Use of this serum ensures the maximal range of induction possible with the Tet Systems. This FBS has been processed in the United States. Documents Components Image Data Back Fetal bovine serum (FBS) approved for use with tetracycline-inducible expression systems Fetal bovine serum (FBS) approved for use with tetracycline inducible expression systems. Only Takara Bio USA functionally tests FBS to be sure that no contaminating tetracycline derivatives interfere with induced expression levels. Back Our fetal bovine serum is functionally tested to guarantee consistent results with tetracycline-inducible expression systems Fetal bovine serum from Clontech is functionally tested to guarantee consistent results with tetracycline-inducible expression systems. Serum from other vendors that have not been functionally tested may contain tetracyclines that affect the maximum expression in Tet-Off systems, or generate background in Tet-On systems. Data here shows induction of luciferase expression from our CHO-AA8-Luc Tet-Off Control Cell Line using different sources of FBS.