[Establishment of Flp-InTM CHO cell lines with double expression of CYP2A13 and MRP2]

Abstract

Objective To construct a double transfected Flp-In^TM CHO cell line stably expressing both cytochrome P450 family 2 subfamily A member 13(CYP2A13) and multidrug resistance-associated protein 2(MRP2). Methods We constructed the recombinant plasmids of pCMV6-NEO-CYP2A13 and pcDNA5-MRP2. The pCMV6-NEO-CYP2A13 recombinant plasmid was first transfected into Flp-In^TM CHO cells, and CYP2A13-Flp-In^TM CHO cells with higher CYP2A13 activity were screened using limiting dilution method and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) cytotoxicity assay. Thereafter, pcDNA5-MRP2 was transfected into CYP2A13-Flp-In^TM CHO cells. The expression levels and activities of CYP2A13 and MRP2 in the double transfected cells and normal cells were detected by real-time quantitative PCR, Western blot analysis and NNK cytotoxicity assay in order to screen Flp-In^TM CHO cells with stable expression of CYP2A13 and MRP2. Results Compared with non-transfected cells, the expression of CYP2A13 and the sensitivity of NNK toxicity in CYP2A13-Flp-In^TM CHO cells increased. The expression of CYP2A13 and MRP2 in CYP2A13/MRP2-Flp-In^TM CHO cells also increased significantly. Compared with CYP2A13-Flp-In^TM CHO cells, CYP2A13/MRP2-Flp-In^TM CHO cells showed no significant difference in CYP2A13 expression; the expression of MRP2 increased while the sensitivity of NNK toxicity decreased significantly. Conclusion The double transfected cell model of CYP2A13 and MRP2 has been successfully established, which lays the foundation for the study of in situ activation of respiratory carcinogens.