Generation Of Novel Model For Studying P-Glycoprotein Based On 3D Organoids And CRISPR/Cas9 System

P-glycoprotein(P-gp)is an efflux transporter localized on the cell membrane that affects the absorption,distribution,metabolism and excretion of drugs.P-gp could significantly decrease the drug bioavailabilities and may lead to tumor multidrug resistance.P-gp-mediated drug transports have attracted much attention in drug research and development,but the present in vitro and in vivo models are not good to mimic the exact processes of P-gp-mediated drug transports in humans.Thus,novel models for studying P-gp are needed.In this study,firstly,we established a novel in vitro model using human small intestinal 3D organoids for studying P-gp-mediated drug transports,especially screening P-gp inhibitors.The small intestinal crypts were separated and then cultured in matrigel with several growth factors.The expression of P-gp in organoids was verified by RT-PCR and immunohistochemistry,and the function of P-gp was verified with Rhodamine123(Rh123)and P-gp inhibitors.We collected the intra-organoid Rh 123 by incubation with PBS,which was efficient and fast.Secondly,we employed the CRISPR/Cas9 system in knocking out rat P-gp and obtained Mdr1a(-/-).Mdrlh(-/-)and Mdr1a(-/-)/1b(-/-)rats which could be well used to study P-gp-mediated drug transports.As the third generation genome-editing tool,CRISPR/Cas9 is widely used to knock out several genes synchronously with many advantages,such as economy,easily obtaining,high precision and no species restriction.sgRNA and Cas9 mRNA were co-injected into the rat embryos and the embryos were transferred to pseudopregnant rats.After two generations of breeding,we obtained P-gp knockout rats,of which Mdr1b(-/-)and Mdr1a(-/-)/1b(-/-)rats were initially constructed.In summary,this study established novel in vitro and in vivo models as useful tools for studying P-gp-mediated drug transports,particularly screening P-gp inhibitors.