Invitrogen™ Flp-In™ T-REx™ Core Kit

• 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