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 output: Contribution to journalArticlepeer-review

18 Citations (Scopus)

Abstract

Purpose To investigate whether it is possible to specifically suppress the expression and function of endogenous canine Pglycoprotein (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 knockdowns 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.

Original languageEnglish
Pages (from-to)2060-2071
Number of pages12
JournalPharmaceutical research
Volume32
Issue number6
DOIs
Publication statusPublished - 2015 Jun 1

Keywords

  • ABC-transporter
  • Breast cancer resistance protein
  • MDCKII
  • P-glycoprotein
  • Zinc finger nucleases

ASJC Scopus subject areas

  • Biotechnology
  • Molecular Medicine
  • Pharmacology
  • Pharmaceutical Science
  • Organic Chemistry
  • Pharmacology (medical)

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