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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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Abstract

Purpose

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.

Methods

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.

Results

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.

Conclusion

Specific efflux mediated by hPGP or hBCRP can be determined with MDCKII cells where cPgp has been knocked out by ZFN.

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Abbreviations

A-B:

Apical-to-basal

ABC–transporters:

ATP-binding cassette transporters

B-A:

Basal-to-apical

cBcrp:

Canine breast cancer resistance protein

cMrp:

Canine multidrug resistance-associated protein

cPgp:

Canine p-glycoprotein

ER:

Efflux ratio

hBCRP:

Human breast cancer resistance protein

hPGP:

Human p-glycoprotein

ko-cell:

cPgp knockout cell

LC-MS/MS:

Liquid chromatography-tandem mass spectrometry

LQ:

The limit of quantification

MDCKII:

Madin-Darby canine kidney type II cell line

NER:

Net efflux ratio

QTAP:

Quantitative targeted absolute proteomics

SRM:

Selected reaction monitoring

ULQ:

Under the limit of quantification

ZFN:

Zinc finger nuclease

ZFP:

Zinc finger protein

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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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Authors and Affiliations

  1. Institute of Pharmacy and Molecular Biotechnology, Ruprecht-Karls-University, Im Neuenheimer Feld 366, 69120, Heidelberg, Germany

    Dominik Gartzke & Gert Fricker

  2. AbbVie Deutschland GmbH & Co. KG, 67061, Ludwigshafen, Germany

    Jürgen Delzer, Loic Laplanche & Jens Sydor

  3. Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, 980-8578, Japan

    Yasuo Uchida, Yutaro Hoshi, Masanori Tachikawa & Tetsuya Terasaki

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  1. Dominik Gartzke
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  2. Jürgen Delzer
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Correspondence to Gert Fricker.

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Gartzke, D., Delzer, J., Laplanche, L. et al. Genomic Knockout of Endogenous Canine P-Glycoprotein in Wild-Type, Human P-Glycoprotein and Human BCRP Transfected MDCKII Cell Lines by Zinc Finger Nucleases. Pharm Res 32, 2060–2071 (2015). https://doi.org/10.1007/s11095-014-1599-5

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  • DOI: https://doi.org/10.1007/s11095-014-1599-5

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