title: Claudin-2 knockout by TALEN-mediated gene targeting in MDCK cells: claudin-2 independently determines the leaky property of tight junctions in MDCK cells.
abstract: Tight junctions (TJs) regulate the movements of substances through the paracellular pathway, and claudins are major determinants of TJ permeability. Claudin-2 forms high conductive cation pores in TJs. The suppression of claudin-2 expression by RNA interference in Madin-Darby canine kidney (MDCK) II cells (a low-resistance strain of MDCK cells) was shown to induce a three-fold increase in transepithelial electrical resistance (TER), which, however, was still lower than in high-resistance strains of MDCK cells. Because RNA interference-mediated knockdown is not complete and only reduces gene function, we considered the possibility that the remaining claudin-2 expression in the knockdown study caused the lower TER in claudin-2 knockdown cells. Therefore, we investigated the effects of claudin-2 knockout in MDCK II cells by establishing claudin-2 knockout clones using transcription activator-like effector nucleases (TALENs), a recently developed genome editing method for gene knockout. Surprisingly, claudin-2 knockout increased TER by more than 50-fold in MDCK II cells, and TER values in these cells (3000-4000 Ω·cm2) were comparable to those in the high-resistance strains of MDCK cells. Claudin-2 re-expression restored the TER of claudin-2 knockout cells dependent upon claudin-2 protein levels. In addition, we investigated the localization of claudin-1, -2, -3, -4, and -7 at TJs between control MDCK cells and their respective knockout cells using their TALENs. Claudin-2 and -7 were less efficiently localized at TJs between control and their knockout cells. Our results indicate that claudin-2 independently determines the 'leaky' property of TJs in MDCK II cells and suggest the importance of knockout analysis in cultured cells.
authors.affiliation: Division of Cell Biology, Department of Physiology and Cell Biology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.
authors.forename: Shinsaku
authors.initials: S
authors.lastname: Tokuda
authors.affiliation: Division of Cell Biology, Department of Physiology and Cell Biology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan; Division of Cerebral Structure, National Institute for Physiological Sciences, Okazaki 444-8787, Japan; Department of Physiological Sciences, The Graduate University for Advanced Studies (SOKENDAI), Okazaki 444-8585, Japan.
authors.forename: Mikio
authors.initials: M
authors.lastname: Furuse
mesh_terms: D000818:Animals; D062446:Claudin-2 / Q000235:genetics / Q000502:physiology*; D003599:Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure; D004285:Dogs; D017097:Electric Impedance; D055786:Gene Knockout Techniques; D018390:Gene Targeting; D061985:Madin Darby Canine Kidney Cells; D010539:Permeability; D034622:RNA Interference; D062725:Tight Junction Proteins / Q000032:analysis / Q000378:metabolism; D019108:Tight Junctions / Q000235:genetics / Q000378:metabolism* / Q000502:physiology
chemical_list: D062446:Claudin-2; D062725:Tight Junction Proteins