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Invitrogen™ T-REx™ Core Kit, with pcDNA™4/TO/myc-His© Vector
Description
Includes
20μg of pcDNA4/TO or 20μg each of pcDNA4/TO/myc-His A, B, and C; a positive control vector, pcDNA6/TR, and forward and reverse sequencing primers. All vectors are supercoiled and provided lyophilized.
- 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 repressor mechanism that blocks transcription from powerful CMV promoter in absence of tetracycline
- Because T-REx System elements do not use viral transactivators, can achieve high-level expression from complete CMV promoter without secondary, non-specific activation of host genes
- Two tetracycline operator sequences (TetO2) have been inserted between TATA box of CMV promoter and transcriptional start site
- TetO2 sequence itself has no effect on expression: When 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 causing TR protein to release TetO2 sites and derepressing transcription from CMV promoter: The 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
- 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 recombinant protein with Anti-myc Antibody and 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 tetracycline repressor (TR) protein
- Vector expresses Blasticidin resistance gene for rapid selection of mammalian cell lines that stably express TR protein
Mammalian Expression, Protein Expression, Proteins, Expression, Isolation and Analysis, Regulated Expression
Specifications
Specifications
| Constitutive or Inducible System | Inducible |
| Delivery Type | Transfection |
| Inducing Agent | Tetracycline |
| Promoter | CMV/TO |
| Product Type | T-Rex Core Cloning Kit with Vector |
| Selection Agent (Eukaryotic) | Zeocin™ |
| Content And Storage | The T-REx™ Core Kit includes 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. |
| Protein Tag | His Tag (6x), c-Myc Epitope Tag |
| Cloning Method | Restriction Enzyme/MCS |
| Quantity | 1 kit |
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Frequently Asked Questions (FAQs)
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.
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.
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.
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).
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.
For Research Use Only. Not for use in diagnostic procedures.