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Invitrogen™ T-REx™ Complete Kit, with pcDNA™4/TO© Vector

Catalog No. K102001
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Catalog No. K102001 Supplier Invitrogen™ Supplier No. K102001
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Description

Includes

20μg of pcDNA4/TO or 20μg each of pcDNA4/TO/myc-His A, B, and C; a positive control vector, pcDNA;6/TR, and forward and reverse sequencing primers plus 1g Zeocin (100mg/mL), 50mg of Blasticidin, and 5g of tetracycline. All vectors are supercoiled and provided lyophilized.

Regulated mammalian expression system yields exceptionally high levels of induced expression

 

  • Uses complete CMV promoter and adds control elements from bacterial tetracycline resistance operon to effectively repress and derepress transcription from one of strongest mammalian promoter sequences known
  • Specific Activation: uses a repressor mechanism that blocks transcription from powerful CMV promoter in absence of tetracycline
  • Because elements do not use viral transactivators, you can achieve high-level expression from complete CMV promoter without secondary, non-specific activation of host genes
  • T-REx transcriptional control elements are provided through two tetracycline operator sequences (TetO2) that have been inserted between TATA box of CMV promoter and transcriptional start site
  • TetO2 sequence itself has no effect on expression
  • When the tetracycline repressor protein (TR) is present, it effectively binds TetO2 sites and blocks transcription initiation
  • Tetracycline added to culture medium binds to, and changes conformation of, TR protein; This change causes TR protein to release TetO2 sites, derepressing transcription from CMV promoter
  • Result is high-level expression of gene of interest
  • Expression levels can be modulated based on tetracycline concentration and can be induced to levels that are achieved with constitutive CMV expression vectors
  • T-REx is a powerful inducible mammalian expression system that allows to regulate expression from complete human cytomegalovirus (CMV) enhancer-promoter
  • Complete CMV enhancer-promoter sequence containing two copies of tetracycline operator TetO2 sequence for high-level regulated expression
  • Zeocinor hygromycin resistance gene for effective selection of stable mammalian cell lines
  • Large multiple cloning site to simplify cloning
  • In addition, pcDNA™4/TO/myc-His offers a c-myc epitope for rapid detection of the recombinant protein with an Anti-myc Antibody and a polyhistidine (6xHis) sequence for simple purification of recombinant protein with nickel-chelating resin and detection with Anti- His(C-term) Antibody
  • Regulatory vector, pcDNA™6/TR, is provided for high-level expression of the tetracycline repressor (TR) protein
  • Expresses Blasticidin resistance gene for rapid selection of mammalian cell lines that stably express the TR protein

 

Mammalian Expression, Protein Expression, Proteins, Expression, Isolation and Analysis, Regulated Expression

Specifications

Constitutive or Inducible System Inducible
Delivery Type Transfection
Inducing Agent Tetracycline
Promoter CMV/TO
Product Type T-Rex Complete Cloning Kit with Vector
Selection Agent (Eukaryotic) Zeocin™, Blasticidin
Content And Storage The T-REx™ Core Kit includes 20 μg of pcDNA™4/TO, a positive control vector, pcDNA™6/TR, and forward and reverse sequencing primers.

The T-REx™ Complete Kit includes the Core Kit plus 1 g Zeocin™ (100 mg/ml), 50 mg of Blasticidin, and 5 g of tetracycline.

All vectors are supercoiled and provided lyophilized.

Store all components at -20°C. Zeocin™ and tetracycline should be protected from exposure to light. All components are guaranteed stable for 6 months when properly stored.
Protein Tag Untagged
Cloning Method Restriction Enzyme/MCS
Quantity 1 kit
Vector pcDNA
Product Line T-REx, pcDNA
For Use With (Application) Regulated Expression
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Frequently Asked Questions (FAQs)

Why is sequential transfection recommended over co-transfection in the T-REx and GeneSwitch systems?

When a co-transfection is performed, there is no way of testing the double stable cell line for functional TetR or GeneSwitch protein, respectively. On the other hand, when sequential transfection is performed, one can functionally test the generated T-REx or GeneSwitch cell line by transiently transfecting the lacZ expression control plasmid and then picking a clone that shows the lowest basal level of expression of lacZ in the absence of the inducer, and the highest level of lacZ in the presence of the inducer. This clone can then be expanded and used to transfect the T-REx or GeneSwitch expression construct, as the case may be.
What is the main advantage of the GeneSwitch system over the T-REx system? And what is its main disadvantage?

With the GeneSwitch system, it is possible to have the absolute lowest basal levels of expression of the gene of interest, whereas the T-REx system may be a little leaky due to the inevitable presence of tetracycline in FBS. The induced level of expression in the GeneSwitch system can be even higher than that seen with the CMV promoter. The disadvantage of the GeneSwitch system is that the expression does not appear to switch off very easily in culture, although it has been demonstrated to function beautifully in transgenics. The T-REx system, on the other hand, can be switched on and off by the addition and removal of the inducer.
What is the advantage of the Flp-In T-REx system over the T-REx system?

The Flp-In T-REx system combines the targeted integration offered by the Flp-In system with the powerful inducible expression offered by the T-REx system. It allows generation of isogenic, inducible, stable cell lines and permits polyclonal selection of these cell lines. Once the Flp-In T-REx host cell line containing an integrated FRT site has been created, subsequent generation of Flp-In T-REx cell lines expressing the gene(s) of interest is rapid and efficient.
Can I use doxycycline instead of tetracycline as an inducer in the T-REx system?

Doxycycline may be used as an alternative inducing agent in the T-REx system. It is similar to tetracycline in its mechanism of action, and exhibits similar dose-response and induction characteristics as tetracycline in the T-REx system. Doxycycline has been shown to have a longer half-life than tetracycline (48 hours vs. 24 hours, respectively). We do not offer doxycycline, but it may be obtained from Sigma (Cat. No. D9891).

I am planning to generate a T-REx cell line using pcDNA6/TR. Can I perform a western blot using antibodies to TetR to assess whether the cell line is expressing enough of TetR? Do you offer an antibody to TetR?

We do not offer an anti-TetR antibody. Even though a western using an anti-TetR antibody can be used to screen out clones that do not express any TetR protein, it would not be the optimal way to screen for functional clones. Functional testing by performing a transient transfection with the lacZ expression control plasmid is recommended for this purpose, followed by picking a clone that shows lowest basal levels of expression of beta-galactosidase in the absence of tetracycline, and highest levels of beta-galactosidase expression upon addition of tetracycline.
I am interested in a mammalian expression system where I can have regulated expression of my gene of interest. I see that you offer multiple systems for this purpose. Can you describe the main features of each system?

We offer three unique mammalian expression systems for inducible/regulated expression of the gene of interest:

- T-REx system
- Flp-In T-REx system
- GeneSwitch system

Please see below to see how they compare with one another:
System -- Basal Expression Level -- Induced Expression Level -- Response time to Maximal Expression -- Transgenic Appliation
T-Rex system -- Low -- Highest -- High -- Suitable
Flp-In T-REx system -- Lower -- High -- 24-48 hrs -- Suitable
GeneSwitch system -- Lowest -- High -- 24-48 hrs -- Suitable
Can the Clontech Tet-On system or Tet-Off system components be used with your T-REx tetracycline-regulated mammalian expression system?

No. The two systems are not compatible since they utilize different strategies for promoter regulation. The T-REx system is designed such that native E. coli tet-repressor protein molecules bind to specific tet-operator sequences (2X TO) just downstream of the TATA box in the full length CMV promoter in the expression vector. This binding keeps the promoter silent simply by preventing the normal transcription machinery from productive assembly at the TATA box. Incidentally, it is this full length CMV promoter region that permits higher induced expression levels relative to other systems.

The recombinant 'repressor' proteins utilized in Clontech's system are actually recombinant fusion proteins which also contain a potent transcriptional transactivator. The Clontech system places operator sequences 5' to the TATA box and relies upon the VP16 transactivator to promote transcription. These repressor-transactivator fusion constructs would have unpredictable and unreliable effects at the CMV promoter in our expression constructs. Additionally, the tet-repressor protein produced from the pCDNA6/TR construct in the T-REx system has no transactivation domain and so would exert little regulatory effect at the minimal promoter region (non-full length CMV) found in the Clontech response plasmids.

For Research Use Only. Not for use in diagnostic procedures.