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

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.

• Authors: Dominik Gartzke, Jürgen Delzer, Loic Laplanche, Yasuo Uchida, Yutaro Hoshi, Masanori Tachikawa, Tetsuya Terasaki, Jens Sydor, Gert Fricker
• Journal: Pharmaceutical research (Pharm Res) Vol. 32 Issue 6 Pg. 2060-71 (Jun 2015) ISSN: 1573-904X [Electronic] United States
• PMID: 25522789 (Publication Type: Journal Article)

Chemical References

• ABCB1 protein, human
• ABCG2 protein, human
• ATP Binding Cassette Transporter, Subfamily B
• ATP Binding Cassette Transporter, Subfamily G, Member 2
• ATP-Binding Cassette Transporters
• Neoplasm Proteins
• RNA, Messenger
• Endodeoxyribonucleases

Topics

• ATP Binding Cassette Transporter, Subfamily B (genetics, metabolism)
• ATP Binding Cassette Transporter, Subfamily G, Member 2
• ATP-Binding Cassette Transporters (genetics, metabolism)
• Animals
• Biological Transport
• Blotting, Western
• DNA Breaks, Double-Stranded
• Dogs
• Endodeoxyribonucleases (genetics, metabolism)
• Gene Expression Regulation
• Gene Knockdown Techniques
• Genotype
• Humans
• Madin Darby Canine Kidney Cells
• Neoplasm Proteins (genetics, metabolism)
• Phenotype
• Proteomics (methods)
• RNA, Messenger (metabolism)
• Species Specificity
• Transfection