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Invitrogen™ Flp-In™ T-REx™ 293 Cell Line

Catalog No. R78007
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R78007 1 mL
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Catalog No. R78007 Supplier Invitrogen™ Supplier No. R78007
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Description

Includes

1 x 107 cells are supplied frozen in 1mL of 90% complete medium and 10% DMSO. Cells must be stored in liquid nitrogen.

Cells contain single stably integrated FRT site at transcriptionally active genomic locus

Mammalian Expression, Mammalian Expression Cell Lines, Protein Expression, Proteins, Expression, Isolation and Analysis, Regulated Expression, Targeted Integration

Specifications

Content And Storage 1 vial of 1 x 107 cells are supplied frozen in 1 ml of 90% complete medium and 10% DMSO. Cells must be stored in liquid nitrogen. Cells are guaranteed stable for 6 months when properly stored.
Cell Line Flp-In T-Rex 293
Species Human
Product Line Flp-In, T-REx
Quantity 1 mL

Frequently Asked Questions (FAQs)

I used one of your T-REx cell lines and am getting expression of my gene of interest in the absence of inducer. Is there any workaround for this problem?

Almost all lots of FBS contain tetracycline, because FBS is generally obtained from cows that have been fed a diet containing tetracycline. If cells are cultured in medium containing FBS that is not reduced in tetracycline, there may be low basal expression of the gene of interest in the absence of added tetracycline. In such cases, we recommend purchasing tetracycline-reduced FBS from our Gibco Cell Culture Division. To be qualified as tetracycline-reduced, these lots must contain below 19.7 ng/mL tetracycline (which happens to be the assay detection limit).

Note: The binding constant for Tet-repressor protein with tetracycline is 3 nM. Assuming that the medium contains 10% serum, a serum tetracycline concentration of 19.7 ng/mL is equivalent to 4 nM tetracycline. Thus, keep in mind that it is possible to get basal level expression even from tetracycline-reduced FBS.

Is multiple integration of the Flp-In expression construct possible? How do you screen for multiple integrants, and how stable is the Flp-In expression cell line?

In theory, one can get multiple integrations of the Flp-In expression construct—an FRT-specific integration event and a random, second-site integration. However, random integration is a relatively uncommon event. Limiting the amount of DNA in the transfection will reduce the chance of second-site integration. We have transfected 293 cells (lacking the FRT site) with the pcDNA5/FRT vector and have identified one potential second-site integrant after screening over 200 clones. DNA integrations can be detected by Southern blot. A single integrant will display a single band; double: two; triple: three, etc. We have maintained a number of Flp-In expression cell lines for over four months and have not observed any loss of the Flp-In expression construct, whether hygromycin selection was maintained or not.
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 kind of Flp-In T-REx cell lines do you offer?

We offer the Flp-In T-REx system that contains pFRT/lacZeo and pcDNA6/TR stably integrated into HEK 293 cells. This cell line has been functionally tested for its ability to regulate expression.
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).

What are the different kinds of T-REx cell lines you offer?

We offer T-REx-293, -HeLa, -CHO, and -Jurkat cell lines. These cell lines are derived by transfection of parental cells with pcDNA6/TR followed by stable selection with blasticidin. They constitutively and stably express the TetR gene, allowing significant time and effort saving when using the T-REx system. These cell lines are functionally tested for expression by transient transfection with the positive control vector, pcDNA4/TO/lacZ. They exhibit extremely low basal expression levels of bGal in the repressed state and high expression upon induction with 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.