EMAIL THIS PAGE TO A FRIEND

Drug metabolism and disposition: the biological fate of chemicals

Zinc finger nuclease-mediated gene knockout results in loss of transport activity for P-glycoprotein, BCRP, and MRP2 in Caco-2 cells.


PMID 25388687

Abstract

Membrane transporters P-glycoprotein [P-gp; multidrug resistance 1 (MDR1)], multidrug resistance-associated protein (MRP) 2, and breast cancer resistance protein (BCRP) affect drug absorption and disposition and can also mediate drug-drug interactions leading to safety/toxicity concerns in the clinic. Challenges arise with interpreting cell-based transporter assays when substrates or inhibitors affect more than one actively expressed transporter and when endogenous or residual transporter activity remains following overexpression or knockdown of a given transporter. The objective of this study was to selectively knock out three drug efflux transporter genes (MDR1, MRP2, and BCRP), both individually as well as in combination, in a subclone of Caco-2 cells (C2BBe1) using zinc finger nuclease technology. The wild-type parent and knockout cell lines were tested for transporter function in Transwell bidirectional assays using probe substrates at 5 or 10 μM for 2 hours at 37°C. P-gp substrates digoxin and erythromycin, BCRP substrates estrone 3-sulfate and nitrofurantoin, and MRP2 substrate 5-(and-6)-carboxy-2',7'-dichlorofluorescein each showed a loss of asymmetric transport in the MDR1, BCRP, and MRP2 knockout cell lines, respectively. Furthermore, transporter interactions were deduced for cimetidine, ranitidine, fexofenadine, and colchicine. Compared with the knockout cell lines, standard transporter inhibitors showed substrate-specific variation in reducing the efflux ratios of the test compounds. These data confirm the generation of a panel of stable Caco-2 cell lines with single or double knockout of human efflux transporter genes and a complete loss of specific transport activity. These cell lines may prove useful in clarifying complex drug-transporter interactions without some of the limitations of current chemical or genetic knockdown approaches.

Related Materials

Product #

Image

Description

Molecular Formula

Add to Cart

MTOX1002P24
BCRP Knockout Caco-2 Cells, one assay ready, 24 well plate
MTOX1002P96
BCRP Knockout Caco-2 Cells, one assay ready, 96-well plate
MTOX1006P24
BCRP/MRP2 Double Knockout Caco-2 Cells, one assay ready, 24 well plate
MTOX1006P96
BCRP/MRP2 Double Knockout Caco-2 Cells, one assay ready, 96-well plate
MTOX1000P24
Caco-2 Control Cells, one assay ready, 24 well plate
MTOX1000P96
Caco-2 Control Cells, one assay ready, 96 well plate
MTOX1001P24
MDR1 Knockout Caco-2 Cells, one assay ready, 24 well plate
MTOX1001P96
MDR1 Knockout Caco-2 Cells, one assay ready, 96-well plate
MTOX1004P24
MDR1/BCRP Double Knockout Caco-2 Cells, one assay ready, 24 well plate
MTOX1004P96
MDR1/BCRP Double Knockout Caco-2 Cells, one assay ready, 96-well plate
MTOX1005P24
MDR1/MRP2 Double Knockout Caco-2 Cells, one assay ready, 24 well plate
MTOX1005P96
MDR1/MRP2 Double Knockout Caco-2 Cells, one assay ready, 96-well plate
MTOX1092P24
MRP1 Knockout Caco-2 Cells, one assay ready, 24 well plate
MTOX1092P96
MRP1 Knockout Caco-2 Cells, one assay ready, 96 well plate
MTOX1003P24
MRP2 Knockout Caco-2 Cells, one assay ready, 24 well plate
MTOX1003P96
MRP2 Knockout Caco-2 Cells, one assay ready, 96-well plate
MTOX1093P24
MRP3 Knockout Caco-2 Cells, one assay ready, 24 well plate
MTOX1093P96
MRP3 Knockout Caco-2 Cells, one assay ready, 96 well plate
MTOX1094P24
MRP4 Knockout Caco-2 Cells, one assay ready, 24 well plate
MTOX1094P96
MRP4 Knockout Caco-2 Cells, one assay ready, 96 well plate
MTOX1095P24
MRP5 Knockout Caco-2 Cells, one assay ready, 24 well plate
MTOX1095P96
MRP5 Knockout Caco-2 Cells, one assay ready, 96 well plate
MTOX1097P24
MRP7 Knockout Caco-2 Cells, one assay ready, 24 well plate
MTOX1097P96
MRP7 Knockout Caco-2 Cells, one assay ready, 96 well plate