Department of Pharmacy, Uppsala University, Uppsala, Sweden; Uppsala University Drug Optimization and Pharmaceutical Profiling Platform, Science for Life Laboratory, Uppsala, Sweden.
Department of Pharmacy, Uppsala University, Uppsala, Sweden.
J Pharm Sci. 2017 Sep;106(9):2909-2913. doi: 10.1016/j.xphs.2017.04.018. Epub 2017 Apr 24.
Madin-Darby canine kidney (MDCK) II cells stably transfected with transport proteins are commonly used models for drug transport studies. However, endogenous expression of especially canine MDR1 (cMDR1) confounds the interpretation of such studies. Here we have established an MDCK cell line stably overexpressing the human MDR1 transporter (hMDR1; P-glycoprotein), and used CRISPR-Cas9 gene editing to knockout the endogenous cMDR1. Genomic screening revealed the generation of a clonal cell line homozygous for a 4-nucleotide deletion in the canine ABCB1 gene leading to a frameshift and a premature stop codon. Knockout of cMDR1 expression was verified by quantitative protein analysis and functional studies showing retained activity of the human MDR1 transporter. Application of this cell line allowed unbiased reclassification of drugs previously defined as both substrates and non-substrates in different studies using commonly used MDCK-MDR1 clones. Our new MDCK-hMDR1 cell line, together with a previously developed control cell line, both with identical deletions in the canine ABCB1 gene and lack of cMDR1 expression represent excellent in vitro tools for use in drug discovery.
稳定转染转运蛋白的犬肾（MDCK）II 细胞常被用作药物转运研究的模型。然而，内源性表达的犬多药耐药基因 1（cMDR1）会干扰此类研究的解释。本研究建立了一株稳定过表达人多药耐药基因 1（hMDR1；P-糖蛋白）的 MDCK 细胞系，并利用 CRISPR-Cas9 基因编辑技术敲除内源性的 cMDR1。基因组筛选显示，该细胞系的一个克隆株在犬 ABCB1 基因中发生了 4 个核苷酸的缺失，导致移码和提前终止密码子。通过定量蛋白分析和功能研究证实 cMDR1 表达被敲除，结果表明人 MDR1 转运体的活性得以保留。该细胞系的应用使得对先前在不同研究中被定义为底物和非底物的药物进行了无偏分类，这些研究中使用了常用的 MDCK-MDR1 克隆株。我们的新 MDCK-hMDR1 细胞系与之前开发的对照细胞系一起，均在犬 ABCB1 基因中缺失相同的序列且不表达 cMDR1，这为药物发现提供了优秀的体外工具。
