T-REx™ Core Kit, with pcDNA™4/TO Vector

Citations & References (4)

Invitrogen™

T-REx™ Core Kit, with pcDNA™4/TO Vector

A Tetracycline-Regulated Expression System without Viral TransactivatorsThe T-REx™ System yields higher levels of induced expression than any other regulated mammalianRead more

Catalog Number	Quantity
K102002 also known as K1020-02	1 kit

Catalog number K102002

also known as K1020-02

Price (MXN)

Quantity:

1 kit

A Tetracycline-Regulated Expression System without Viral Transactivators
The T-REx™ System yields higher levels of induced expression than any other regulated mammalian expression system. It utilizes the complete CMV promoter and adds control elements from the bacterial tetracycline resistance operon to effectively repress and derepress transcription from one of the strongest mammalian promoter sequences known (1,2).

Specific Activation
The T-REx™ System uses a repressor mechanism that blocks transcription from the powerful CMV promoter in the absence of tetracycline. Because the T-REx™ System elements do not use viral transactivators, you can achieve high-level expression from the complete CMV promoter without secondary, non-specific activation of host genes.

The T-REx™ Mechanism
The T-REx™ transcriptional control elements are illustrated in Figure 1. Two tetracycline operator sequences (TetO₂) have been inserted between the TATA box of the CMV promoter and the transcriptional start site. The TetO₂ sequence itself has no effect on expression. When the tetracycline repressor protein (TR) is present, it effectively binds the TetO₂ sites and blocks transcription initiation. Tetracycline added to the culture medium binds to, and changes the conformation of, the TR protein. This change causes the TR protein to release the TetO₂ sites, derepressing transcription from the CMV promoter. The result is high-level expression of the gene of interest (Figure 2). Expression levels can be modulated based on the 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 you to regulate expression from the complete human cytomegalovirus (CMV) enhancer-promoter. T-REx™inducible expression vectors offer the following features:

• Complete CMV enhancer-promoter sequence containing two copies of the tetracycline operator TetO₂ sequence for high-level regulated expression
• Zeocin™ or 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 the recombinant protein with nickel-chelating resin and detection with Anti- His(C-term) Antibody.

The regulatory vector, pcDNA™6/TR, is provided for high-level expression of the tetracycline repressor (TR) protein. This vector expresses the Blasticidin resistance gene for rapid selection of mammalian cell lines that stably express the TR protein.

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

Specifications

Constitutive or Inducible SystemInducible

Delivery TypeTransfection

For Use With (Application)Regulated Expression

Inducing AgentTetracycline

Product TypeT-Rex Core Cloning Kit with Vector

Quantity1 kit

Selection Agent (Eukaryotic)Zeocin™, Blasticidin

VectorpcDNA

Cloning MethodRestriction Enzyme/MCS

Product LineGateway, T-REx, pcDNA

PromoterCMV/TO

Protein TagUntagged

Unit SizeEach

Contents & Storage

The T-REx™ Core Kit includes 20 μg of pcDNA™4/TO or 20 μg each of pcDNA™4/TO/myc-His A, B, and C; a positive control vector, pcDNA™6/TR, and forward and reverse sequencing primers.

All vectors are supercoiled and provided lyophilized.

Store all components at -20°C. All components are guaranteed stable for 6 months when properly stored.

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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

View all T-REx™ Core Kit, with pcDNA™4/TO Vector FAQs

Citations & References (4)

Citations & References

Abstract

Comparison of seven different heterologous protein expression systems for the production of the serotonin transporter.

Authors:Tate CG, Haase J, Baker C, Boorsma M, Magnani F, Vallis Y, Williams DC,

Journal:Biochim Biophys Acta

PubMed ID:12586388

'The rat serotonin transporter (rSERT) is an N-glycosylated integral membrane protein with 12 transmembrane regions; the N-glycans improve the ability of the SERT polypeptide chain to fold into a functional transporter, but they are not required for the transmembrane transport of serotonin per se. In order to define the best ... More

Effect of some aldoses on growth of Saccharomyces cerevisiae inhibited with molybdenum.

Authors:Zemek J, Bílik V, Zákutná L,

Journal:Folia Microbiol (Praha)

PubMed ID:285

The inhibitory effect of molybdenum ions on growth of yeasts at pH 5.5 was found to be decreased by aldoses in the following order: D-talose greater than L-mannose greater than L-ribose greater than D-lyxose greater than L-galactose greater than L-arabinose greater than L-glucose greater than L-xylose. Increased concentrations of molybdenum ... More

Human TRPC5 channel activated by a multiplicity of signals in a single cell.

Authors:Zeng F, Xu SZ, Jackson PK, McHugh D, Kumar B, Fountain SJ, Beech DJ,

Journal:J Physiol

PubMed ID:15254149

Here we explore the activation mechanisms of human TRPC5, a putative cationic channel that was cloned from a region of the X chromosome associated with mental retardation. No basal activity was evident but activity was induced by carbachol stimulation of muscarinic receptors independently of Ca2+ release. This is 'receptor activation', ... More

The gamma -secretase-cleaved C-terminal fragment of amyloid precursor protein mediates signaling to the nucleus.

Authors: Gao Y; Pimplikar S W;

Journal:Proc Natl Acad Sci U S A

PubMed ID:11742091

Sequential processing of the amyloid precursor protein (APP) by beta- and gamma-secretases generates the Abeta peptide, a major constituent of the senile plaques observed in Alzheimer's disease. The cleavage by gamma-secretase also results in the cytoplasmic release of a 59- or 57-residue-long C-terminal fragment (Cgamma). This processing resembles regulated intramembrane ... More