Generation and characterisation of an organotypic cell knockout model to study pulmonary MRP1
Johannes A. Sake1, Lyubomyr Burtnyak2, Mohammed Ali Selo1,3, Severin Mairinger4,5, Henriette E. Dähnhardt1, Camelia Helbet1, Vincent P. Kelly2, Oliver Langer4,5, Carsten Ehrhardt1
1School of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin, Dublin, Ireland
2School of Biochemistry and Immunology, Trinity College Dublin, Dublin, Ireland
3Faculty of Pharmacy, University of Kufa, AL-Najaf, Iraq
4Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria
5Department of Biomedical Imaging and Image-guided Therapy, Medical University of Vienna, Vienna, Austria
Summary
We recently reported a contribution of multidrug resistance-associated protein 1 (MRP1/ABCC1) to pulmonary drug absorption in vivo. To study pulmonary MRP1 function in vitro, an organotypic knockout model based on the human NCI-H441 distal lung epithelial cell line was generated and characterised.
A CRISPR/Cas9 technique using the knock-in/promoter trap method was employed. Pre-assembled spCas9 protein/gRNA construct and repair template were delivered to NCI-H441 cells by electroporation. Repair templates contained puromycin resistance and mCherry fluorescence genes. The successful knock-in was evaluated by puromycin selection and mCherry detection via flow cytometry. In the thus generated clones ABCC1 gene and MRP1 protein expression were compared to wild-type (WT) cells by real-time polymerase chain reaction (qPCR) and immunoblot, respectively. Transporter activity was studied using 6-bromo-7-methylpurine (BMP), a prodrug of the MRP1-specific substrate S-(6-(7-methylpurinyl))glutathione (MPG).
Initially, eight KO clones (M1–M8) were generated, of which only M1 and M2 showed significantly (p ≤ 0.001) reduced ABCC1 mRNA levels, while MRP1 protein was undetectable in all clones. Transport studies in M2 revealed significantly (p ≤ 0.001) reduced release of MPG compared to WT cells. The inhibitory effect was comparable to the MRP1 inhibitor MK-571.
We successfully generated MRP1 KO clones, which can be used to study the potential influence of MRP1 on pulmonary drug disposition and physiology on mechanistic levels in vitro.
Key Message
The efflux transporter MRP1 is a major contributor to the extrusion of xenobiotics and endogenous substances from pulmonary epithelial cells. The organotypic knockout model based on NCI-H441 cells generated in this study offers the opportunity to study MRP1 activity in vitro, which was previously not possible due to unspecific pharmacological substrates/inhibitors.