Thermo Fisher, K650001, Flp-In™ T-REx™ Core Kit

Quantity: 1 kit
Unit Size: Each
• 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 thelacZ-Zeocin fusion
• Latter constitutively expresses tetracycline repressor, which will enable to regulate activity of TetO_2promoter
• 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/TetO_2promoter, 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 eventMammalian Expression, Protein Expression, Proteins, Expression, Isolation and Analysis, Regulated Expression, Targeted Integration