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Characterisation of endogenous Claudin‐1 expression, motility and susceptibility to hepatitis C virus in CRISPR knock‐in cells
Author(s) -
Clément Camille M.H.,
Deffieu Maika S.,
Dorobantu Cristina M.,
Baumert Thomas F.,
AyalaNunez Nilda Vanesa,
Mély Yves,
Ronde Philippe,
Gaudin Raphael
Publication year - 2020
Publication title -
biology of the cell
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.543
H-Index - 85
eISSN - 1768-322X
pISSN - 0248-4900
DOI - 10.1111/boc.201900085
Subject(s) - biology , claudin , microbiology and biotechnology , cell culture , gene knockin , endogeny , fluorescence recovery after photobleaching , cell , tight junction , genetics , gene , biochemistry , membrane
Background Information Claudin‐1 (CLDN1) is a four‐span transmembrane protein localised at cell–cell tight junctions (TJs), playing an important role in epithelial impermeability and tissue homoeostasis under physiological conditions. Moreover, CLDN1 expression is up‐regulated in several cancers, and the level of CLDN1 expression has been proposed as a prognostic marker of patient survival. Results Here, we generated and characterised a novel reporter cell line expressing endogenous fluorescent levels of CLDN‐1, allowing dynamic monitoring of CLDN‐1 expression levels. Specifically, a hepatocellular carcinoma Huh7.5.1 monoclonal cell line was bioengineered using CRISPR/Cas9 to endogenously express a fluorescent TagRFP‐T protein fused at the N‐terminus of the CLDN1 protein. These cells were proved useful to measure CLDN1 expression and distribution in live cells. However, the cells were resistant to hepatitis C virus (HCV) infection, of which CLDN1 is a viral receptor, while retaining permissiveness to VSV‐G‐decorated pseudoparticles. Nonetheless, the TagRFP‐CLDN1 +/+ cell line showed expected CLDN1 protein localisation at TJs and the cell monolayer had similar impermeability and polarisation features as its wild‐type counterpart. Finally, using fluorescence recovery after photobleaching (FRAP) approaches, we measured that the majority of endogenous and overexpressed TagRFP‐CLDN1 diffuses rapidly within the TJ, whereas half of the overexpressed EGFP‐CLDN1 proteins were stalled at TJs. Conclusions The Huh7.5.1 TagRFP‐CLDN1 +/+ edited cell line showed physiological features comparable to that of non‐edited cells, but became resistant to HCV infection. Our data also highlight the important impact of the fluorescent protein chosen for endogenous tagging. Significance Although HCV‐related studies may not be achieved with these cells, our work provides a novel tool to study the cell biology of TJ‐associated proteins and a potential screening strategy measuring CLDN1 expression levels.