Genomic Knockout of Endogenous Canine P-Glycoprotein in Wild-Type, Human P-Glycoprotein and Human BCRP Transfected MDCKII Cell Lines by Zinc Finger Nucleases
| Autor: | Masanori Tachikawa, Yasuo Uchida, Dominik Gartzke, Gert Fricker, Tetsuya Terasaki, Yutaro Hoshi, Jens Sydor, Loic Laplanche, Jürgen Delzer |
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| Rok vydání: | 2014 |
| Předmět: |
Proteomics
ATP Binding Cassette Transporter Subfamily B Genotype Abcg2 Blotting Western Cell Pharmaceutical Science Transfection Madin Darby Canine Kidney Cells Dogs Species Specificity medicine ATP Binding Cassette Transporter Subfamily G Member 2 Animals Humans DNA Breaks Double-Stranded Pharmacology (medical) RNA Messenger P-glycoprotein Pharmacology Endodeoxyribonucleases biology fungi Organic Chemistry Wild type Biological Transport Zinc finger nuclease Molecular biology Neoplasm Proteins Blot Phenotype medicine.anatomical_structure Gene Expression Regulation Cell culture Gene Knockdown Techniques biology.protein Molecular Medicine ATP-Binding Cassette Transporters Biotechnology |
| Zdroj: | Pharmaceutical Research. 32:2060-2071 |
| ISSN: | 1573-904X 0724-8741 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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