Citation

  • Authors: Clement CMH. et al.
  • Year: 2020
  • Journal: Biol Cell 112 140-151
  • Applications: in vitro / DNA / jetPRIME
  • Cell type: Huh-7.5.1
    Description: Human hepatocarcinoma cells
    Known as: Huh7,5,1, Huh 7,5,1

Method

70% confluency at the time of transfection, 6 well plate, 700 ng of each : donor plasmid, gRNA-tracrRNA coding plasmid, Streptococcus pyogenes Cas9, 0.2% GFP positive cells then cell were sorted to obtain a pool of 70% GFP positive.

Abstract

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.Keywords: CLDN1; FRAP; Gene editing; HCV; Hepatocellular carcinoma.

Pubmed