Invitrogen™ Flp-In™ T-REx™ Core Kit

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

Brand:  Invitrogen™ K650001

Product Code. 10173712

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

Description

• 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
• Inducing Agent: Tetracycline
• Product Type: Flp-In Core Kit
• 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.
• Cloning Method: Restriction Enzyme/MCS
• Vector: pFRT
• For Use With (Application): Regulated Expression

• Delivery Type: Transfection
• Promoter: CMV/TO
• Selection Agent (Eukaryotic): Zeocin™, Blasticidin, Hygromycin
• Protein Tag: Untagged
• Quantity: 1 kit
• Product Line: Flp-In™, T-REx™

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