Abstract A series of stable breast cancer resistance protein (BCRP, ABCG2) knockdown cell lines were produced by transduction of Caco-2 cells with lentiviral vector-based shRNA. Caco-2 cell is a human intestinal-derived cell line widely used to study intestinal drug absorption. Caco-2 expresses three apical drug efflux transporters: BCRP, P-glycoprotein (P-gp; ABCB1) and multidrug resistance protein 2 (MRP2, ABCC2). BCRP and P-gp, in particular, play a significant role in pharmacokinetics due to their expression at several key interfaces. Over-expression of BCRP in cancer cells may also be a mechanism of tumor resistance to chemotherapeutic drugs. The goal of this study was to engineer and characterize Caco-2 cell clones with stable knockdown of BCRP expression. shRNA/BCRP lentiviral particles were used to infect a stable clone of Caco-2 cells. Expression of BCRP was monitored using quantitative PCR (qPCR), western blotting, immunofluorescence microscopy and bidirectional transport of probe substrates, estrone-3-sulfate (E3S) and pheophorbide A (PhA). Based on qPCR, expression of BCRP mRNA was knocked down in five clones with a maximum of 97% silencing in Clone D. Silencing of BCRP gene expression was maintained for at least 25 passages. Expression of BCRP protein was also reduced significantly. Functionally, BCRP knockdown was reflected in significant reduction of the efflux ratio of E3S and PhA. Clone D in particular should be a useful model for identifying and characterizing P-gp substrates and inhibitors without interference from BCRP and/or MRP2. In addition, it can be used in conjunction with wild-type or vector control Caco-2 cells to identify BCRP substrates. DMD 23309
