Caco-2 Transporter Knockout Cell-Based Assays

Service Cell Line Transporter Technology Membrane Type Probe Substrate Read Out Reference Inhibitor Positive Control Dynamic Range
Caco-2 BCRP substrate assessment / Transporter knockout Caco-2-C2BBe1-BCRP-KO* BCRP
Monolayer Assay
Estrone 3-sulfate (E3S) Ko143
Caco-2 MDR1/BCRP substrate assessment / Transporter double knockout Caco-2-C2BBe1-MDR1/BCRP-KO* BCRP
Monolayer Assay
Digoxin, Estrone 3-sulfate (E3S) Valspodar or Ko143
Caco-2 MDR1/MRP2 substrate assessment / Transporter double knockout Caco-2-C2BBe1-MDR1/MRP2-KO* MDR1/P-gp
Monolayer Assay
CDCF-DA, Digoxin Valspodar or MK-571
Caco-2 MRP2/BCRP substrate assessment / Transporter double knockout Caco-2-C2BBe1-MRP2/BCRP-KO* BCRP
Monolayer Assay
Estrone 3-sulfate (E3S), CDCF-DA MK-571 or Ko143
Caco-2 MRP2 substrate assessment / Transporter knockout Caco-2-C2BBe1-MRP2-KO* MRP2
Monolayer Assay
Caco-2 MDR1 substrate assessment / Transporter knockout Caco-2-C2BBe1-MDR1-KO* MDR1/P-gp
Monolayer Assay
Digoxin Valspodar

*These assays utilize the parental Caco-2-C2BBe1 cell line as a positive control of transporter-specific function. Transport of the test article is compared between parental and transporter-knockout Caco-2-C2BBe1 cells. A chemical inhibitor can be added optionally to the control cell.


Substrate assessment with efflux transporter knockout cells

SOLVO expanded its monolayer substrate assay portfolio with transporter specific Caco2 (C2BBe1 subclone) knockout cells. Transporter knockout Caco2 cells are a good method for determining the potential impact of specific efflux transporters in the intestine on the absorption and disposition of a drug.

Transporter knockout cell lines can be used to identify specific drug-transporter interactions by comparison of drug transport between the wild-type (WT) and the knockout cell lines, without dependence on chemical inhibitors. Transporter specific knockout strains of the Caco2 cell line were developed by Sigma-Aldrich (Sigma, St Louis MO). Functional knock out Caco2 cells were developed by utilizing CompoZr® Zinc Finger Nucleases (ZFNs) [1,2]. The resultant MDR1, BCRP and MRP2 knockout cells demonstrated disruption of gene sequence as well as complete loss of transporter function in bidirectional transport assays out to at least 40 passages post-ZFN genomic modification.

These cell lines have been characterized at SOLVO and proven to be an alternative tool for elucidating drug-transporter interactions. Model compounds, known to be substrates for these efflux transporters such as: digoxin, estrone 3-sulfate, and CDCF have been used, and in each case, the efflux ratio of the compound decreases close to unity in the appropriate knockout cell line versus the WT cell line. Characterization data is available upon request.

Substrate assessment assay setup


1. Carroll D (2011) Genetics 188; 773-782
2. Pratt, J et al. , (2012) Current Protocols in Toxicology 52. 23.2.1-23.2.22