Takara Lenti-X Tet-One Inducible Expression System (Puro) \Each

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 thePTRE3GSpromoter 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.

Tet-One System Mechanism — The Tet-On 3G transactivator protein is expressed constitutively from the humanPGKpromoter(PPGK) but is unable to bind to theTRE3Gpromoter(PTRE3G) in the absence of doxycycline (Dox).When bound by Dox, supplied in the culture medium, the transactivator undergoes a conformational change, binds toPTRE3Gand activates transcription of a transgene cloned downstream.

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).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.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).

Required Products

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.

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.

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.

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.

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.

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.