Genomic Knockout of Endogenous Canine P-Glycoprotein in Wild-Type, Human P-Glycoprotein and Human BCRP Transfected MDCKII Cell Lines by Zinc Finger Nucleases
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To investigate whether it is possible to specifically suppress the expression and function of endogenous canine P-glycoprotein (cPgp) in Madin-Darby canine kidney type II cells (MDCKII) transfected with hPGP and breast cancer resistance protein (hBCRP) by zinc finger nuclease (ZFN) producing sequence specific DNA double strand breaks.
Wild-type, hPGP-transfected, and hBCRP-transfected MDCKII cells were transfected with ZFN targeting for cPgp. Net efflux ratios (NER) of Pgp and Bcrp substrates were determined by dividing efflux ratios (basal-to-apical / apical-to-basal) in over-expressing cell monolayers by those in wild-type ones.
From ZFN-transfected cells, cell populations (ko-cells) showing knockout of cPgp were selected based on genotyping by PCR. qRT-PCR analysis showed the significant knock-downs of cPgp and interestingly also cMrp2 expressions. Specific knock-downs of protein expression for cPgp were shown by western blotting and quantitative targeted absolute proteomics. Endogenous canine Bcrp proteins were not detected. For PGP-transfected cells, NERs of 5 Pgp substrates in ko-cells were significantly greater than those in parental cells not transfected with ZFN. Similar result was obtained for BCRP-transfected cells with a dual Pgp and Bcrp substrate.
Specific efflux mediated by hPGP or hBCRP can be determined with MDCKII cells where cPgp has been knocked out by ZFN.
KEY WORDSABC–transporter breast cancer resistance protein MDCKII P-glycoprotein zinc finger nucleases
ATP-binding cassette transporters
Canine breast cancer resistance protein
Canine multidrug resistance-associated protein
Human breast cancer resistance protein
cPgp knockout cell
Liquid chromatography-tandem mass spectrometry
The limit of quantification
Madin-Darby canine kidney type II cell line
Net efflux ratio
Quantitative targeted absolute proteomics
Selected reaction monitoring
Under the limit of quantification
Zinc finger nuclease
Zinc finger protein
ACKNOWLEDGMENTS AND DISCLOSURES
The authors would like to thank Dr. Axel Meyer for his suggestions on how to construct the zinc finger nucleases.
Tetsuya Terasaki is a full professor at Tohoku University, and is also a director of Proteomedix Frontiers Co. Ltd. This study was not supported by Proteomedix Frontiers Co. Ltd., and his position at Proteomedix Frontiers Co. Ltd. did not affect the design of the study, the collection of the data, the analysis or interpretation of the data, the decision to submit the manuscript for publication, or the writing of the manuscript and did not present any financial conflicts. The other authors declare no competing interests.
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