Construction of HEK293 cells stably expressing wild-type organic anion transporting polypeptide 1B1 (OATP1B1☆1a) and variant OATP1B1☆1b and OATP1B1☆15

A transgenic cell line stably expressing the human organic anion transporting polypeptide (OATP1B1) was established. Human Embryonic Kidney 293 (HEK293) cell line stably expressing OATP1B1(star)1a sequence was amplified through PCR with the extracted total RNA as templates from human liver, then subcloned into the plasmid pMD19-T and verified by sequencing. OATP1B1(star)1b/OATP1B1(star)15 mutant sequences were obtained by site-directed mutation PCR with pMD19-T/OATP1B1(star)1a as templates. The plasmids pcDNA3.1(+)/OATP1B1(star)1a, (star)1b and (star)15 were constructed and transfected into HEK293 cell line using Lipofectannine (TM) 2000 transfection reagent. Several stable transfected clones were obtained after selection with G418. Using rosuvastatin as a probe substrate of OATP1B1, the intracellular rosuvastatin accumulation in HEK293 and HEK-OATP1B1(star)1a, (star)1b and (star)15 monoclone cells were validated by a ultra-performance liquid chromatography-tandem mass spectrometry. OATP1B1 mRNA and protein expression were detected by RT-PCR and Western blot, respectively. The results from RT-PCR, rosuvastatin uptake and Western blot assay indicated that human OATP1B1 was highly expressed in transfected cells compared with controls. The HEK-293 cell lines stably expressing human OATP1B1-wild and variant (HEK-OATP1B1, (star)1b and (star)15) are potential models to study drug transport in vitro.