Flp-In™ T-REx™ Core Kit

Name: Flp-In™ T-REx™ Core Kit
SKU: K650001
Price: 2785.00 USD

The Flp-In™ T-REx™ System allows the generation of stable mammalian cell lines exhibiting tetracycline-inducible expression of a gene of interestRead more

Catalog number K650001

Price (USD)

2,785.00

Each

Add to cart

Price (USD)

2,785.00

Each

Add to cart

The Flp-In™ T-REx™ System allows the generation of stable mammalian cell lines exhibiting tetracycline-inducible expression of a gene of interest from a specific genomic location.

Features of the Flp-In™ T-REx™ System include:

• 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.
• The Flp-In™ T-REx™ System allows the generation of isogenic, inducible stable cell lines.
• The Flp-In™ T-REx™ System permits polyclonal selection of stable expression cell lines.

Create One Host Cell Line for Multiple Expression Applications
To generate your Flp-In™ T-REx™ host cell line for the expression of your gene, you will first sequentially transfect a mammalian cell line with pFRT/lacZeo and pcDNA6/TR, both of which integrate randomly and independently into the host genome. The former contains an FRT site (for homologous intermolecular recombination) just downstream from the initiation codon of the lacZ-Zeocin™ fusion. The latter constitutively expresses the tetracycline repressor, which will enable you to regulate the activity of the TetO₂ promoter.

Targeted Integration and Regulated Expression
The next step in generating your inducible cell line is to co-transfect the pOG44 and pcDNA5/FRT/TO vectors, the latter of which contains your gene of interest under the control of a tetracycline-regulated CMV/TetO₂ promoter, into your host cell line. Homologous recombination between FRT sites in pcDNA5/FRT/TO and on the host cell chromosome, catalyzed by the Flp recombinase expressed from pOG44, generates the derivative cell lines into which your gene has been stably integrated into the host cell chromosome. These cells can be selected and maintained using the hygromycin and blasticicin resistance acquired as a consequence of the recombination event.

For research use only. Not for use in diagnostic procedures.

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

Specifications

Constitutive or Inducible SystemInducible

Delivery TypeTransfection

Inducing AgentTetracycline

PromoterCMV/TO

Product TypeFlp-In Core Kit

Selection Agent (Eukaryotic)Zeocin™, Blasticidin, Hygromycin

Protein TagUntagged

Cloning MethodRestriction Enzyme/MCS

Quantity1 kit

VectorpFRT

Product LineFlp-In™, T-REx™

For Use With (Application)Regulated Expression

Unit SizeEach

Contents & 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.

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.

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

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.

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

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