missing translation for 'onlineSavingsMsg'
Learn More
  1. Home
  2. Products
  3. Molecular Biology Reagents and Kits
  4. DNA Vectors
  5. K650001

Invitrogen™ Flp-In™ T-REx™ Core Kit

Catalog No. K650001
Encompass
Change view
Click to view available options
Quantity:
1 kit
1 product options available for selection
Product selection table with 1 available options. Use arrow keys to navigate and Enter or Space to select.
Catalog No. Quantity
K650001 1 kit
Use arrow keys to navigate between rows. Press Enter or Space to select a product option. 1 options available.
1 options
Catalog No. K650001 Supplier Invitrogen™ Supplier No. K650001
Only null left

Description

Includes

pFRT/lacZeo: 20μg, lyophilized in TE, pcDNA6/TR: 20μg, lyophilized in TE, pOG44: 20μg, lyophilized in TE, pcDNA5/FRT/TO: 20μg, lyophilized in TE, pcDNA5/FRT/TO/CAT: 20μg, lyophilized in TE, CMV Forward Primer (21-mer): 2μg, lyophilized in TE, BGH Reverse Primer (18-mer): 2μg, lyophilized in TE, Tetracycline: 5g

Allows generation of stable mammalian cell lines exhibiting tetracycline-inducible expression of gene of interest from specific genomic location

  • Once Flp-InT-REx host cell line containing integrated FRT site has been created, subsequent generation of Flp-InT-REx cell lines expressing gene(s) of interest is rapid and efficient
  • Allows generation of isogenic, inducible stable cell lines
  • Permits polyclonal selection of stable expression cell lines
  • To generate Flp-InT-REx host cell line for expression of gene, will first sequentially transfect mammalian cell line with pFRT/lacZeo and pcDNA6/TR, both of which integrate randomly and independently into host genome
  • Former contains FRT site (for homologous intermolecular recombination) just downstream from initiation codon of the lacZ-Zeocin fusion
  • Latter constitutively expresses tetracycline repressor, which will enable to regulate activity of TetO2 promoter
  • Next step in generating inducible cell line is to co-transfect the pOG44 and pcDNA5/FRT/TO vectors, latter of which contains gene of interest under control of tetracycline-regulated CMV/TetO2 promoter, into host cell line
  • Homologous recombination between FRT sites in pcDNA5/FRT/TO and on host cell chromosome, catalyzed by Flp recombinase expressed from pOG44, generates derivative cell lines into which gene has been stably integrated into host cell chromosome
  • These cells can be selected and maintained using hygromycin and blasticicin resistance acquired as consequence of recombination event

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

Specifications

Constitutive or Inducible System Inducible
Delivery Type Transfection
Inducing Agent Tetracycline
Promoter CMV/TO
Product Type Flp-In Core Kit
Selection Agent (Eukaryotic) Zeocin™, Blasticidin, Hygromycin
Content And Storage Each kit contains the following components:
• pFRT/lacZeo: 20 μg, lyophilized in TE
• pcDNA6/TR: 20 μg, lyophilized in TE
• pOG44: 20 μg, lyophilized in TE
• pcDNA5/FRT/TO: 20 μg, lyophilized in TE
• pcDNA5/FRT/TO/CAT: 20 μg, lyophilized in TE
• CMV Forward Primer (21-mer): 2 μg, lyophilized in TE
• BGH Reverse Primer (18-mer): 2 μg, lyophilized in TE
• Tetracycline: 5 g
The kit ships at room temperature. Store vectors and primers at -20°C. Store tetracycline at 4°C, protected from light.
Protein Tag Untagged
Cloning Method Restriction Enzyme/MCS
Quantity 1 kit
Vector pFRT
Product Line Flp-In, T-REx
For Use With (Application) Regulated Expression
Show More Show Less

Frequently Asked Questions (FAQs)

I performed the Flp-In reaction and obtained hygromycin-resistant clones which also turned out to be Zeocin antibiotic resistant and lacZ-positive. I am concerned that the Flp-In reaction has not worked. Can you explain what might be happening?

We have observed in-house that in cells where the FRT site has integrated into a very transcriptionally active locus in the host cell genome (seen more commonly in Flp-In CHO and Flp-In 293 cells but can also happen in Flp-In 3T3 cells and any other Flp-In host cell line), there is some "read-through" transcription and translation of the lacZ-Zeocin ORF subsequent to the Flp-In reaction, even though the lacZ-Zeocin ORF does not have a bonafide promoter and ATG. In such cases, the hygromycin-resistant clones would also be lacZ-positive and Zeocin antibiotic-resistant. To make sure that the integration is FRT site-specific and not random, we recommend doing a parallel control transfection with no pOG44 present. This should yield no surviving clones upon hygromycin selection, indicating that all the hygromycin-resistant clones obtained in the presence of pOG44 are indeed Flp recombinase-dependent and hence have the gene of interest integrated at the FRT site. Also, a Southern blot analysis of these clones will help verify that they do indeed have proper FRT integration of the gene of interest despite the expression of lacZ (although this is typically not necessary). After the Flp-In reaction, as long as you see hygromycin-resistant clones, we recommend that you select them and assay them for expression of your gene of interest.

I used the pcDNA5/FRT vector in your Flp-In 3T3 cell line and was initially able to express my gene of interest, but after a month or so, lost expression. Why is this?

The Flp-In 3T3 cell line is derived from NIH3T3 cells, which are mouse fibroblast cells. The CMV promoter is known to get silenced over time in murine cell lines and hence we would recommend using a Flp-In expression vector with a non-CMV promoter in these cells, such as the pEF5/FRT/V5-D-TOPO vector or the pEF5/FRT/V5-DEST vector.
I used the pFRT/lacZeo vector to generate my own Flp-In host cell line and then made my Flp-In expression cell line. I am seeing very poor expression of my gene of interest. Is there anything else I can try to improve expression?

Before giving up, we would suggest that you try using the pFRT/lacZeo2 vector to generate your host cell line. This vector contains a truncated version of the SV40 promoter driving the lacZ-Zeocin fusion. Use of this vector facilitates the isolation of clones that have integrated the vector near enhancer elements in the genome, thus resulting in higher levels of expression of the gene of interest.
What is the difference between the Jump-In and Flp-In systems?

The Jump-In system is PhiC31-integrase mediated and is a stable, targeted, and irreversible mammalian expression system. It consists of the Jump-In Fast system that involves a single integration step and the Jump-InTI (targeted integration) system that needs two integration steps, both of which are targeted and irreversible. In contrast, the Flp-In system is a stable, targeted mammalian expression system that is reversible. The first integration is random (integration of pFRT/lacZeo), and the second integration (integration of the Flp-In expression vector) is targeted but reversible.
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 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 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
When should I consider reversible integration (Flp-In system) vs irreversible integration (Jump-In system)?

Use irreversible integration (Jump-In system) if the transgene should be sustained in the mammalian genome for a long time. Use reversible integration such as Flp-In system if the transgene needs to be replaced with another gene of interest after a short period of time.
What is the difference between Jump-In and Flp-In systems?

The Jump-In system is PhiC31-integrase mediated and is a stable, targeted, and irreversible mammalian expression system, involving one integration step. The Jump-In TI (Targeted Integration) system needs two integration steps, both of which are targeted and irreversible. In contrast, the Flp-In system is a stable, targeted mammalian expression system that is reversible. The first integration is random (integration of pFRT/lacZeo) and the second integration (integration of the Flp-In expression vector) is targeted but reversible.
In the Flp-In system, how can one explain the presence of hygromycin-resistant clones that are also resistant to Zeocin antibiotic and are lacZ positive?

We have observed in-house that in cells where the FRT site has integrated into a very transcriptionally active locus in the host cell genome (seen more commonly in Flp-In CHO and Flp-In 293 cells but can also happen in Flp-In 3T3 cells and any other Flp-In host cell line), there is some "read-through" transcription and translation of the lacZ-Zeocin ORF post Flp-In recombination, even though the lacZ-Zeocin ORF does not have a bona fide promoter and ATG. In such cases, the hygromycin-resistant clones would also be lacZ positive and Zeocinantibiotic-resistant. To make sure that the integration is FRT site-specific and not random, we recommend doing a parallel control transfection with no pOG44 present. This should yield no surviving clones upon hygromycin selection, indicating that all the hygromycin-resistant clones obtained in the presence of pOG44 are indeed Flp recombinase-dependent and hence have the gene of interest integrated at the FRT site. Also, a Southern blot analysis of these clones will help verify that they do indeed have proper FRT integration of the gene of interest despite the expression of lacZ (although this is typically not necessary). Post Flp-Inrecombination, as long as you see hygromycin-resistant clones, we recommend that you select them and assay them for expression of your gene of interest.
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.