Establishment of HEK293 cell line expressing green fluorescent protein-aquaporin-1 to determine osmotic water permeability.
Aquaporin (AQP) is a kind of channel-forming membrane glycoprotein that mediates osmotic water transport. The present study aimed to establish a cell line stably transfected with AQP1 to measure osmotic water permeability. The recombinant plasmid was constructed by subcloning the full-length rat AQP1 cDNA into pEGFP-C3 vector, named pEGFP/AQP1. Human embryonic kidney 293 cells were transfected with pEGFP/AQP1 and selected by G418 to obtain a cell line stably expressing AQP1 tagged with green fluorescent protein. The expression level of AQP1 in the stably transfected cell was detected by reverse transcription polymerase chain reaction and Western blot. The real-time change of fluorescence density, corresponding to cell swelling induced by hyposmotic solution, was recorded under confocal laser scanning microscope and used to assess osmotic water permeability. The typical AQP1 inhibitor, mercuric chloride, validated this osmotic water permeability assay. These results suggested that this transfected cell model could be conveniently used to determine osmotic water permeability.
Gao
Junwei
J
Department of Pharmacology and State Key Laboratory of Natural & Biomimetic Drugs, Peking University Health Science Center, Beijing 100083, China.
Yu
Heming
H
Song
Qianliu
Q
Li
Xuejun
X
D051398:Aquaporin 1; D020346:Aquaporins / Q000096:biosynthesis* / Q000235:genetics; D001789:Blood Group Antigens; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D007668:Kidney / Q000196:embryology; D018613:Microscopy, Confocal; D009995:Osmosis / Q000502:physiology*; D014162:Transfection / Q000379:methods; D014867:Water / Q000502:physiology*
C496020:AQP1 protein, human; D020346:Aquaporins; D001789:Blood Group Antigens; D014867:Water; D051398:Aquaporin 1; D049452:Green Fluorescent Proteins