Establishment of Flp-In CHO recombinant cell lines stably expressing CYP2A13 and CYP2A13/POR
- Author:
Li SUN
1
;
Huan LIU
1
;
Jun-Qi HE
1
;
Jia SUN
1
;
Yong-Lin WANG
1
;
Chang YANG
1
;
Ting LIU
1
;
Li SUN
2
;
Huan LIU
2
;
Jun-Qi HE
2
;
Ding-Yan LU
3
;
Yong-Jun LI
3
Author Information
- Publication Type:Journal Article
- Keywords: cytochrome P450 family 2 subfamily A member 13; cytochrome P450 oxidoreductase; Flp-In CHO cell; metabolic activity; pre-carcinogens; stable expression
- From: Chinese Pharmacological Bulletin 2022;38(5):795-799
- CountryChina
- Language:Chinese
- Abstract: Aim To construct Flp-In CHO cell line(CYP2A13-CHO)stably expressing cytochrome P450 family 2 subfamily A member 13(CYP2A13)and Flp-In CHO cell line(CYP2A13-POR-CHO)stably co-expressing CYP2A13 and cytochrome P450 oxidoreductase(POR), from which a cell line with better metabolic activity is selected.Method In our previous study, we had constructed a Flp-In CHO cell line(POR-Flp-In CHO)stably expressing POR using lentiviral vector.The recombinant plasmids of pcDNA5/FRT-CYP2A13 were constructed and transfected into Flp-In CHO cells and POR-Flp-In CHO cells through LipofectamineTM 2000.The expression and activity of CYP2A13 were detected by real-time quantitative PCR(qRT-PCR), Western blot and Aflatoxin B1(AFB1)/4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK)cytotoxicity assay and the metabolic activity was compared between CYP2A13-CHO and CYP2A13-POR-CHO.Results Compared with non-transfected cells, the mRNA and protein expression of CYP2A13 in CYP2A13-CHO and CYP2A13-POR-CHO cells both increased significantly.Besides, compared with CYP2A13-POR-CHO, CYP2A13-CHO cells were more sensitive to AFB1 and NNK.Conclusions The Flp-In CHO cell line stably expressing CYP2A13 and with better metabolic activity has been established successfully, which provides a tool for screening of pre-carcinogens that can be metabolically activated by CYP2A13.
