Contruction And Biologocal Indentification Of Double-transfected MDCKâ…¡ Cell Line Expressing HMDR1and HBCRP

Objective To clone human BCRP coding region gene and construct a plasmid expressingstably hBCRP gene, and construct an engineered MDCKII-hMDR1/hBCRP cell line whichconstantly express the human BCRP and MDR1gene, and identify and study of theirboilogical function.Method Human BCRP total RNA was isolated from MDCKII-hBCRP cells by Trizol rea-gent. The isolated mRNA was reverse-transcripted to synthesize the first cDNA, which wasused as the template for hBCRP amplification by PCR. Then, it was inserted intomammalian expression vector pcDNA3.1/Hygro to construct recombinant vector pcDNA3.1/Hygro/hBCRP．The recombinant vector was transfected into MDCKII-hMDR1cellswith PolyJet DNA In vitro Transfection reagent，and cell clones stably expressing humanBCRP molecule were screened by Hygromycin B．We used RT-PCR to verify expressionof hBCRP from mRNA level.Transwell system was used to identify and study of P-gp and BCRP biological functionin MDCKII-hMDR1/hBCRP cell lines. A Shimadzu HPLC system and Agela Venusil C18column (2.1mm50mm,5μm) were used in the separation of probe substrates and theirrespective internal standards. The analytes were eluted using a linear gradient with mobilephase A (Acetonitrile: water: formic acid,5/95/0.1, v/v) and mobile phase B (Acetonitrile:water:formic acid,95/5/0.1, v/v).AB API4000mass detection was carried out byelectrospray ionization (ESI) in multiple reaction monitoring (MRM) mode with iontransitions of m/z329.4â†'m/z162.5for Labetolol, m/z313.0â†'m/z270.1for Dantrolene,m/z494.5â†'m/z394.2for Imatinib and m/z465.1â†'m/z270.1for Sorafenib, and theirrespective internal standards with ion transitions of m/z285.0â†'m/z193.2for Dizeapam,m/z391.3â†'m/z361.4for Betamethasone, m/z488.3â†'m/z232.3for Dasatinib and m/z 393.3â†'m/z349.3for SN-38.Results We have constructed successfully the mammalian transfer vector pcDNA3.1/Hygro/hBCRP and screened for the engineered cell line MDCKII-hMDR1/hBCRP thatconstantly express the human MDR1gene．Both transporter proteins were functionallyactive.Conclusion We have constructed successfully a MDCKII-hMDR1/hBCRP cell lineco-expressing hMDR1and hBCRP gene. Using these double-transfectants, we identifiednew substrates transcelluar transported sequentially by P-gp and BCRP. And it will be as auseful in vitro model to study the interplay of P-gp and BCRP in the Blood Brain Barrier(BBB).