[Construction of Flp-InTM CHO cell line stably expressing human cytochrome P450 oxidoreductase].

Abstract

Objective To establish a Flp-In^TM CHO cell line stably expressing human cytochrome P450 oxidoreductase (POR) to lay a solid foundation for the further construction of cell lines stably co-expressing human POR and human cytochrome P450 (CYP). Methods POR recombinant lentivirus was established and infected with Flp-In^TM CHO cells, and the expression of green fluorescent protein was observed by fluorescence microscope for monoclonal screening. Mitomycin C (MMC) cytotoxic assay, Western blot analysis and quantitative real-time PCR (qRT-PCR) were employed to detect the activity and expression of POR, and eventually obtained a cell line stably expressing POR (Flp-In^TM CHO-POR). Flp-In^TM CHO-POR cells (Flp-In^TM CHO-POR-2C19) stably co-expressing POR and CYP2C19, and Flp-In^TM CHO cells stably expressing CYP2C19 (Flp-In^TM CHO-2C19) were constructed, and the activity of CYP2C19 was measured by cyclophosphamide (CPA). Results The consequences of MMC cytotoxic assay, Western blot and qRT-PCR illuminated that Flp-In^TM CHO cells infected with POR recombinant lentivirus had elevated MMC metabolic activity and boosted the expression of POR mRNA and protein, compared with Flp-In^TM CHO cells infected with negative control virus, indicating that Flp-In^TM CHO-POR cells stably expressing POR were obtained. No significant disparity existed in the metabolic activity of CPA between Flp-In^TM CHO-2C19 and Flp-In^TM CHO cells, whereas the metabolic activity enhanced in Flp-In^TM CHO-POR-2C19 and was significantly higher than in Flp-In^TM CHO-2C19 cells. Conclusion The stable expression of Flp-In^TM CHO-POR cell line is successfully established and can be further applied to construct CYP transgenic cells.