Claudin-2 knockout by TALEN-mediated gene targeting in MDCK cells: claudin-2 independently determines the leaky property of tight junctions in MDCK cells.

Shinsaku Tokuda,Mikio Furuse,Christopher R. Weber

doi:10.1371/journal.pone.0119869

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.

Highlights

In multicellular organisms, epithelia act as a barrier to the external environment
The transcription activator-like effector nucleases (TALENs) constructs were transfected into Madin-Darby canine kidney (MDCK) II cells, and immunofluorescence analysis of claudin-2 revealed complete loss of claudin-2 staining at cell-cell contacts in some regions, indicating the validity of the TALEN constructs for claudin-2 gene knockout in MDCK II cells (Fig. 1B)
To increase the efficiency of the selection of knockout clones, we cloned a pair of TALEN DNA constructs for claudin-2 knockout into mammalian expression vectors with neomycin- and puromycin-resistance genes

Summary

Introduction

Epithelia act as a barrier to the external environment. Epithelial cells adhere to each other through complexes that form junctions between the cells, and the tight junction (TJ) is located in the most apical part of the complexes [1]. TJs regulate the movement of substances through paracellular pathways of the various permeabilities found among epithelia (barrier function), contributing to the generation and maintenance of the proper internal environment required for organ function [2,3]. The exogenous expression of claudin-2 in Madin-Darby canine kidney (MDCK) I cells, a high-resistance strain of MDCK cells that lack claudin-2 expression, decreased transepithelial electrical resistance (TER), a reciprocal of the ion conductance across the epithelia, by more than 10-fold, and transformed so-called ‘tight’ epithelia into ‘leaky’ epithelia [9,10]. The suppression of claudin-2 expression by RNA interference (knockdown) in MDCK II cells, a low-resistance strain of MDCK cells that express endogenous claudin-2, induced a three-fold increase in TER [13,14]. The values of TER in claudin-2 knockdown MDCK II cells were markedly lower (130–250 OÁcm2) than those in high-resistance strains of MDCK cells (> 1000 OÁcm). Because RNA interference-mediated knockdown is not complete and only reduces gene function, another possibility is that claudin-2 pores formed from residual claudin-2 expression during knockdown could still have significant effects on TER, resulting in lower TER in claudin-2 knockdown cells

Methods

Results

Conclusion