*4.4. Stable Expression of ABCB1 in HEK293 Flp-in Cells*

HEK293 Flp-In cells with stable expression of ABCB1 wild-type were generated previously [10]. The cDNAs encoding ABCB1-Q347/990A and ABCB1-Q347/725/990A were excised from their parent pCI-neo plasmids using BamHI/NotI restriction endonuclease double digests and subcloned into the equivalent sites of pcDNA5/FRT (ThermoFisher Sci, Waltham, MA, USA). The resulting pcDNA5/FRT-ABCB1-Q347/990A and pcDNA5/FRT-ABCB1-Q347/725/990A (Qtriple) were used to co-transfect HEK293 Flp-In cells (ThermoFisher Sci, Waltham, MA, USA) along with the pOG44 (ThermoFisher Sci, Waltham, MA, USA) as a source of Flp recombinase as described by the manufacturer. Stable transfected cells (Flp-In ABCB1-Q347/990A, Flp-In ABCB1-Q347/725/990A (Qtriple) and pcDNA/FRT as a vector-only negative control) were selected with hygromycin (200 µg/mL) and, once uniform expression of the mutant ABCB1 were confirmed, maintained in hygromycin (100 µg/mL).
