Flp-In™ T-REx™ Core Kit

Catalog number: K650001

Invitrogen™  Related applications: Protein Expression

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Description

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

Promoter: CMV⁄TO
Key Function: Regulated Expression
Protein Tag or Fusion: Untagged
Delivery Method: Transfection
Cloning Method: Restriction Enzyme ⁄ MCS
Selection Agent (Eukaryotic): Blasticidin, Hygromycin, Zeocin™
Vector Type: pFRT
Product Size: 1 kit
Inducing Agent: Tetracycline
Constitutive or Inducible System: Inducible

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.

Documents

Manuals & protocols