title: Genomic Knockout of Endogenous Canine P-Glycoprotein in Wild-Type, Human P-Glycoprotein and Human BCRP Transfected MDCKII Cell Lines by Zinc Finger Nucleases.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  Specific efflux mediated by hPGP or hBCRP can be determined with MDCKII cells where cPgp has been knocked out by ZFN.
authors.affiliation: Institute of Pharmacy and Molecular Biotechnology, Ruprecht-Karls-University, Im Neuenheimer Feld 366, 69120, Heidelberg, Germany.
authors.forename: Dominik
authors.initials: D
authors.lastname: Gartzke
authors.forename: Jürgen
authors.initials: J
authors.lastname: Delzer
authors.forename: Loic
authors.initials: L
authors.lastname: Laplanche
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Uchida
authors.forename: Yutaro
authors.initials: Y
authors.lastname: Hoshi
authors.forename: Masanori
authors.initials: M
authors.lastname: Tachikawa
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Terasaki
authors.forename: Jens
authors.initials: J
authors.lastname: Sydor
authors.forename: Gert
authors.initials: G
authors.lastname: Fricker
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000235:genetics / Q000378:metabolism; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D053903:DNA Breaks, Double-Stranded; D004285:Dogs; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D005838:Genotype; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D040901:Proteomics / Q000379:methods; D012333:RNA, Messenger / Q000378:metabolism; D013045:Species Specificity; D014162:Transfection
chemical_list: C513055:ABCB1 protein, human; C116614:ABCG2 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D004706:Endodeoxyribonucleases