title: Construction of HEK293 cells stably expressing wild-type organic anion transporting polypeptide 1B1 (OATP1B1*1a) and variant OATP1B1*1b and OATP1B1*15.
abstract: 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*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*1b/OATP1B1*15 mutant sequences were obtained by site-directed mutation PCR with pMD19-T/ OATP1B1*1a as templates. The plasmids pcDNA3.1(+)/OATP1B1*1a, *1b and *15 were constructed and transfected into HEK293 cell line using Lipofectamine 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*1a, *1b and *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, *1b and *15) are potential models to study drug transport in vitro.
authors.forename: M
authors.initials: M
authors.lastname: Chen
authors.forename: B X
authors.initials: BX
authors.lastname: Qu
authors.forename: X L
authors.initials: XL
authors.lastname: Chen
authors.forename: H H
authors.initials: HH
authors.lastname: Hu
authors.forename: H D
authors.initials: HD
authors.lastname: Jiang
authors.forename: L S
authors.initials: LS
authors.lastname: Yu
authors.forename: Q
authors.initials: Q
authors.lastname: Zhou
authors.forename: S
authors.initials: S
authors.lastname: Zeng
mesh_terms: D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D014644:Genetic Variation; D057809:HEK293 Cells / Q000378:metabolism*; D006801:Humans; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000378:metabolism; D008055:Lipids; D008099:Liver / Q000378:metabolism; D027381:Liver-Specific Organic Anion Transporter 1; D016297:Mutagenesis, Site-Directed; D027361:Organic Anion Transporters / Q000096:biosynthesis* / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D000068718:Rosuvastatin Calcium / Q000378:metabolism; D014162:Transfection / Q000379:methods*
chemical_list: D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D008055:Lipids; C086724:Lipofectamine; D027381:Liver-Specific Organic Anion Transporter 1; D027361:Organic Anion Transporters; D012333:RNA, Messenger; C503999:SLCO1B1 protein, human; D000068718:Rosuvastatin Calcium