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



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.


ABC–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


Canine p-glycoprotein


Efflux ratio


Human breast cancer resistance protein


Human p-glycoprotein


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



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.

Supplementary material

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


  1. 1.
    Cho MJ, Thompson DP, Cramer CT, Vidmar TJ, Scieszka JF. The Madin Darby Canine Kidney (MDCK) epithelial cell monolayer as a model cellular transport barrier. Pharm Res. 1989;6(1):71–7.CrossRefPubMedGoogle Scholar
  2. 2.
    Irvine JD, Takahashi L, Lockhart K, Cheong J, Tolan JW, Selick HE, et al. MDCK (Madin-Darby Canine Kidney) cells: a tool for membrane permeability screening. J Pharm Sci. 1999;88(1):28–33.CrossRefPubMedGoogle Scholar
  3. 3.
    Braun A, Hammerle S, Suda K, Rothen-Rutishauser B, Gunthert M, Kramer SD, et al. Cell cultures as tools in biopharmacy. Eur J Pharm Sci. 2000;11 Suppl 2:S51–60.CrossRefPubMedGoogle Scholar
  4. 4.
    Yamazaki M, Neway WE, Ohe T, Chen I, Rowe JF, Hochman JH, et al. In vitro substrate identification studies for p-glycoprotein-mediated transport: species difference and predictability of in vivo results. J Pharmacol Exp Ther. 2001;296(3):723–35.PubMedGoogle Scholar
  5. 5.
    Pastan I, Gottesman MM, Ueda K, Lovelace E, Rutherford AV, Willingham MC. A retrovirus carrying an MDR1 cDNA confers multidrug resistance and polarized expression of P-glycoprotein in MDCK cells. Proc Natl Acad Sci U S A. 1988;85(12):4486–90.CrossRefPubMedCentralPubMedGoogle Scholar
  6. 6.
    Horio M, Chin KV, Currier SJ, Goldenberg S, Williams C, Pastan I, et al. Transepithelial transport of drugs by the multidrug transporter in cultured Madin-Darby canine kidney cell epithelia. J Biol Chem. 1989;264(25):14880–4.PubMedGoogle Scholar
  7. 7.
    Polli JW, Wring SA, Humphreys JE, Huang L, Morgan JB, Webster LO, et al. Rational use of in vitro P-glycoprotein assays in drug discovery. J Pharmacol Exp Ther. 2001;299(2):620–8.PubMedGoogle Scholar
  8. 8.
    Goh LB, Spears KJ, Yao D, Ayrton A, Morgan P, Roland Wolf C, et al. Endogenous drug transporters in in vitro and in vivo models for the prediction of drug disposition in man. Biochem Pharmacol. 2002;64(11):1569–78.CrossRefPubMedGoogle Scholar
  9. 9.
    Gartzke D, Fricker G. Establishment of optimized MDCK cell lines for reliable efflux transport studies. J Pharm Sci. 2014;103(4):1298–304.CrossRefPubMedGoogle Scholar
  10. 10.
    Kim YG, Cha J, Chandrasegaran S. Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci U S A. 1996;93(3):1156–60.CrossRefPubMedCentralPubMedGoogle Scholar
  11. 11.
    Smith J, Berg JM, Chandrasegaran S. A detailed study of the substrate specificity of a chimeric restriction enzyme. Nucleic Acids Res. 1999;27(2):674–81.CrossRefPubMedCentralPubMedGoogle Scholar
  12. 12.
    Klug A, Schwabe JW. Protein motifs 5. Zinc fingers. FASEB J. 1995;9(8):597–604.PubMedGoogle Scholar
  13. 13.
    Urnov FD, Miller JC, Lee YL, Beausejour CM, Rock JM, Augustus S, et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature. 2005;435(7042):646–51.CrossRefPubMedGoogle Scholar
  14. 14.
    Bibikova M, Beumer K, Trautman JK, Carroll D. Enhancing gene targeting with designed zinc finger nucleases. Science. 2003;300(5620):764.CrossRefPubMedGoogle Scholar
  15. 15.
    Porteus MH, Baltimore D. Chimeric nucleases stimulate gene targeting in human cells. Science. 2003;300(5620):763.CrossRefPubMedGoogle Scholar
  16. 16.
    Beumer K, Bhattacharyya G, Bibikova M, Trautman JK, Carroll D. Efficient gene targeting in Drosophila with zinc-finger nucleases. Genetics. 2006;172(4):2391–403.CrossRefPubMedCentralPubMedGoogle Scholar
  17. 17.
    Moehle EA, Rock JM, Lee YL, Jouvenot Y, DeKelver RC, Gregory PD, et al. Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases. Proc Natl Acad Sci U S A. 2007;104(9):3055–60.CrossRefPubMedCentralPubMedGoogle Scholar
  18. 18.
    Vanamee ES, Santagata S, Aggarwal AK. FokI requires two specific DNA sites for cleavage. J Mol Biol. 2001;309(1):69–78.CrossRefPubMedGoogle Scholar
  19. 19.
    Bitinaite J, Wah DA, Aggarwal AK, Schildkraut I. FokI dimerization is required for DNA cleavage. Proc Natl Acad Sci U S A. 1998;95(18):10570–5.CrossRefPubMedCentralPubMedGoogle Scholar
  20. 20.
    Smith J, Bibikova M, Whitby FG, Reddy AR, Chandrasegaran S, Carroll D. Requirements for double-strand cleavage by chimeric restriction enzymes with zinc finger DNA-recognition domains. Nucleic Acids Res. 2000;28(17):3361–9.CrossRefPubMedCentralPubMedGoogle Scholar
  21. 21.
    Hauschild-Quintern J, Petersen B, Cost GJ, Niemann H. Gene knockout and knockin by zinc-finger nucleases: current status and perspectives. Cell Mol Life Sci. 2013;70(16):2969–83.CrossRefPubMedGoogle Scholar
  22. 22.
    Hafez M, Hausner G. Homing endonucleases: DNA scissors on a mission. Genome. 2012;55(8):553–69.CrossRefPubMedGoogle Scholar
  23. 23.
    Tachibana T, Kitamura S, Kato M, Mitsui T, Shirasaka Y, Yamashita S, et al. Model analysis of the concentration-dependent permeability of P-gp substrates. Pharm Res. 2010;27(3):442–6.CrossRefPubMedGoogle Scholar
  24. 24.
    Kamiie J, Ohtsuki S, Iwase R, Ohmine K, Katsukura Y, Yanai K, et al. Quantitative atlas of membrane transporter proteins: development and application of a highly sensitive simultaneous LC/MS/MS method combined with novel in-silico peptide selection criteria. Pharm Res. 2008;25(6):1469–83.CrossRefPubMedGoogle Scholar
  25. 25.
    Kuteykin-Teplyakov K, Luna-Tortos C, Ambroziak K, Loscher W. Differences in the expression of endogenous efflux transporters in MDR1-transfected versus wildtype cell lines affect P-glycoprotein mediated drug transport. Br J Pharmacol. 2010;160(6):1453–63.CrossRefPubMedCentralPubMedGoogle Scholar
  26. 26.
    Hoshi Y, Uchida Y, Tachikawa M, Inoue T, Ohtsuki S, Terasaki T. Quantitative atlas of blood-brain barrier transporters, receptors, and tight junction proteins in rats and common marmoset. J Pharm Sci. 2013;102(9):3343–55.CrossRefPubMedGoogle Scholar
  27. 27.
    Uchida Y, Tachikawa M, Obuchi W, Hoshi Y, Tomioka Y, Ohtsuki S, et al. A study protocol for quantitative targeted absolute proteomics (QTAP) by LC-MS/MS: application for inter-strain differences in protein expression levels of transporters, receptors, claudin-5, and marker proteins at the blood-brain barrier in ddY, FVB, and C57BL/6J mice. Fluids Barriers CNS. 2013;10(1):21.CrossRefPubMedCentralPubMedGoogle Scholar
  28. 28.
    Uchida Y, Ohtsuki S, Katsukura Y, Ikeda C, Suzuki T, Kamiie J, et al. Quantitative targeted absolute proteomics of human blood-brain barrier transporters and receptors. J Neurochem. 2011;117(2):333–45.CrossRefPubMedGoogle Scholar
  29. 29.
    Shawahna R, Uchida Y, Decleves X, Ohtsuki S, Yousif S, Dauchy S, et al. Transcriptomic and quantitative proteomic analysis of transporters and drug metabolizing enzymes in freshly isolated human brain microvessels. Mol Pharm. 2011;8(4):1332–41.CrossRefPubMedGoogle Scholar
  30. 30.
    Feng B, Mills JB, Davidson RE, Mireles RJ, Janiszewski JS, Troutman MD, et al. In vitro P-glycoprotein assays to predict the in vivo interactions of P-glycoprotein with drugs in the central nervous system. Drug Metab Dispos. 2008;36(2):268–75.CrossRefPubMedGoogle Scholar
  31. 31.
    Martin SE, Caplen NJ. Applications of RNA interference in mammalian systems. Annu Rev Genomics Hum Genet. 2007;8:81–108.CrossRefPubMedGoogle Scholar
  32. 32.
    Choudhuri S, Klaassen CD. Structure, function, expression, genomic organization, and single nucleotide polymorphisms of human ABCB1 (MDR1), ABCC (MRP), and ABCG2 (BCRP) efflux transporters. Int J Toxicol. 2006;25(4):231–59.CrossRefPubMedGoogle Scholar
  33. 33.
    Wang D, Johnson AD, Papp AC, Kroetz DL, Sadee W. Multidrug resistance polypeptide 1 (MDR1, ABCB1) variant 3435C > T affects mRNA stability. Pharmacogenet Genomics. 2005;15(10):693–704.CrossRefPubMedGoogle Scholar
  34. 34.
    Terzi M. Chromosomal variation and the origin of drug-resistant mutants in mammalian cell lines. Proc Natl Acad Sci U S A. 1974;71(12):5027–31.CrossRefPubMedCentralPubMedGoogle Scholar
  35. 35.
    Hastings PJ, Lupski JR, Rosenberg SM, Ira G. Mechanisms of change in gene copy number. Nat Rev Genet. 2009;10(8):551–64.CrossRefPubMedCentralPubMedGoogle Scholar
  36. 36.
    O’Huallachain M, Karczewski KJ, Weissman SM, Urban AE, Snyder MP. Extensive genetic variation in somatic human tissues. Proc Natl Acad Sci U S A. 2012;109(44):18018–23.CrossRefPubMedCentralPubMedGoogle Scholar
  37. 37.
    Dukes JD, Whitley P, Chalmers AD. The MDCK variety pack: choosing the right strain. BMC Cell Biol. 2011;12:43.CrossRefPubMedCentralPubMedGoogle Scholar
  38. 38.
    Szakacs G, Paterson JK, Ludwig JA, Booth-Genthe C, Gottesman MM. Targeting multidrug resistance in cancer. Nat Rev Drug Discov. 2006;5(3):219–34.CrossRefPubMedGoogle Scholar
  39. 39.
    Silverman JA. Multidrug-resistance transporters. Pharm Biotechnol. 1999;12:353–86.CrossRefPubMedGoogle Scholar
  40. 40.
    Keogh JP, Kunta JR. Development, validation and utility of an in vitro technique for assessment of potential clinical drug-drug interactions involving P-glycoprotein. Eur J Pharm Sci. 2006;27(5):543–54.CrossRefPubMedGoogle Scholar
  41. 41.
    Siarheyeva A, Lopez JJ, Glaubitz C. Localization of multidrug transporter substrates within model membranes. Biochemistry. 2006;45(19):6203–11.CrossRefPubMedGoogle Scholar
  42. 42.
    Veau C, Faivre L, Tardivel S, Soursac M, Banide H, Lacour B, et al. Effect of interleukin-2 on intestinal P-glycoprotein expression and functionality in mice. J Pharmacol Exp Ther. 2002;302(2):742–50.CrossRefPubMedGoogle Scholar
  43. 43.
    Giacomini KM, Huang SM, Tweedie DJ, Benet LZ, Brouwer KL, Chu X, et al. Membrane transporters in drug development. Nat Rev Drug Discov. 2010;9(3):215–36.CrossRefPubMedGoogle Scholar
  44. 44.
    Yamagishi T, Sahni S, Sharp DM, Arvind A, Jansson PJ, Richardson DR. P-glycoprotein mediates drug resistance via a novel mechanism involving lysosomal sequestration. J Biol Chem. 2013;288(44):31761–71.CrossRefPubMedCentralPubMedGoogle Scholar
  45. 45.
    Evans DC, O’Connor D, Lake BG, Evers R, Allen C, Hargreaves R. Eletriptan metabolism by human hepatic CYP450 enzymes and transport by human P-glycoprotein. Drug Metab Dispos. 2003;31(7):861–9.CrossRefPubMedGoogle Scholar
  46. 46.
    Kageyama M, Namiki H, Fukushima H, Ito Y, Shibata N, Takada K. In vivo effects of cyclosporin A and ketoconazole on the pharmacokinetics of representative substrates for P-glycoprotein and cytochrome P450 (CYP) 3A in rats. Biol Pharm Bull. 2005;28(2):316–22.CrossRefPubMedGoogle Scholar
  47. 47.
    Haslam IS, Jones K, Coleman T, Simmons NL. Induction of P-glycoprotein expression and function in human intestinal epithelial cells (T84). Biochem Pharmacol. 2008;76(7):850–61.CrossRefPubMedGoogle Scholar
  48. 48.
    Zhang Y, Laterra J, Pomper MG. Hedgehog pathway inhibitor HhAntag691 is a potent inhibitor of ABCG2/BCRP and ABCB1/Pgp. Neoplasia. 2009;11(1):96–101.CrossRefPubMedCentralPubMedGoogle Scholar
  49. 49.
    Roy U, Chakravarty G, Honer Zu Bentrup K, Mondal D. Montelukast is a potent and durable inhibitor of multidrug resistance protein 2-mediated efflux of taxol and saquinavir. Biol Pharm Bull. 2009;32(12):2002–9.Google Scholar
  50. 50.
    Tai LM, Loughlin AJ, Male DK, Romero IA. P-glycoprotein and breast cancer resistance protein restrict apical-to-basolateral permeability of human brain endothelium to amyloid-beta. J Cereb Blood Flow Metab. 2009;29(6):1079–83.CrossRefPubMedGoogle Scholar
  51. 51.
    de Vries NA, Zhao J, Kroon E, Buckle T, Beijnen JH, van Tellingen O. P-glycoprotein and breast cancer resistance protein: two dominant transporters working together in limiting the brain penetration of topotecan. Clin Cancer Res. 2007;13(21):6440–9.CrossRefPubMedGoogle Scholar
  52. 52.
    Ito K, Uchida Y, Ohtsuki S, Aizawa S, Kawakami H, Katsukura Y, et al. Quantitative membrane protein expression at the blood-brain barrier of adult and younger cynomolgus monkeys. J Pharm Sci. 2011;100(9):3939–50.CrossRefPubMedGoogle Scholar

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

Personalised recommendations