Pharmaceutical Research

, Volume 32, Issue 6, pp 2060–2071 | Cite as

Genomic Knockout of Endogenous Canine P-Glycoprotein in Wild-Type, Human P-Glycoprotein and Human BCRP Transfected MDCKII Cell Lines by Zinc Finger Nucleases

  • Dominik Gartzke
  • Jürgen Delzer
  • Loic Laplanche
  • Yasuo Uchida
  • Yutaro Hoshi
  • Masanori Tachikawa
  • Tetsuya Terasaki
  • Jens Sydor
  • Gert Fricker
Research Paper

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.

KEY WORDS

ABC–transporter breast cancer resistance protein MDCKII P-glycoprotein zinc finger nucleases 

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

Supplementary material

11095_2014_1599_MOESM1_ESM.docx (19 kb)
ESM 1(DOCX 18 kb)

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Dominik Gartzke
    • 1
  • Jürgen Delzer
    • 2
  • Loic Laplanche
    • 2
  • Yasuo Uchida
    • 3
  • Yutaro Hoshi
    • 3
  • Masanori Tachikawa
    • 3
  • Tetsuya Terasaki
    • 3
  • Jens Sydor
    • 2
  • Gert Fricker
    • 1
  1. 1.Institute of Pharmacy and Molecular BiotechnologyRuprecht-Karls-UniversityHeidelbergGermany
  2. 2.AbbVie Deutschland GmbH & Co. KGLudwigshafenGermany
  3. 3.Graduate School of Pharmaceutical SciencesTohoku UniversitySendaiJapan

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