Sigma^® Life Science has applied our exclusive CompoZr Zinc Finger Nuclease (ZFN) technology towards the creation of functional knockouts of intestinal efflux transporters, MDR1, BCRP, and MRP2, for use in identifying drug-drug interactions involving drug transporters (Figure 1).

Future transporter knockout development projects will include a broader range of transporters, including OATP1B1, OATP1B3, OAT1, OAT3, OCT2, BSEP and MATE1, in a variety of base cell types, including liver, kidney and blood brain barrier models.

Figure 1 Through comparison of wildtype cells with CompoZr ZFN created transporter knockout cell lines, interactions between transporters and drug compounds can be explicitly identified.

Assay Benefits

• Utilize true knockout of key drug transporter(s) eliminating the reliance on nonspecific substrates and/or inhibitors in drug-transporter studies
• Explicitly identify the efflux transporter(s) responsible for poor absorption of new compounds
• Insert directly into your standard drug transporter testing funnel
• Caco2-derived cell line (C2BBe1)
• Validated using industry-standard bidirectional transport assay

Functional Data

Genotype Analysis of BCRP Knockout

Homozygous knockout (no functional protein)

• 4 base pair deletion
• 5 base pair insertion
• WT: AGGAGATCAGCTACACCACCTCCTTCTGTcatcaACTCAGATGGGTTTCCAAGCGTTC
• Allele 1&2: AGGAGATCAGCTACACCACCTCCTTCTGT----aACTCAGATGGGTTTCCAAGCGTTC
• Allele 3&4: AGGAGATCAGCTACACCACCTCCTTCTGTcGTCATatcaACTCAGATGGGTTTCCAAGCGTTC

Figure 2: The transport of Digoxin, CDCFDA, and Estrone-3-Sulfone (E3S) in C2BBe1 BCRP knockout cells. 
The Efflux Ratio (B-A/A-B) of the MDR1 substrate, Digoxin, the MRP2 substrate, CDCFDA, and the BCRP substrate, Estrone-3-Sulfone (E3S), was measured in the C2BBe1 BCRP knockout cell line and the wildtype C2BBe1 cell line. As can be seen in the Figure below, the efflux ratio for E3S is eliminated in the BCRP knockout cell line, whereas, the efflux ratio for digoxin and CDCFDA remains unaffected.

If you are interested in discussing developmental partnership opportunities for transporter focused assays or other areas of interest with Sigma Life Science, please contact us at admetox@sial.com.

Or fill out the ADME/Tox Partnership Request Form.

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