Development and characterization of a unified in vitro model system for comparative analysis of the GJB2 gene variants associated with hearing loss

Functional analysis of mutational changes in genes in model systems in vitro plays a key role in understanding the molecular mechanisms of hereditary pathologies and is an important criterion for confirmation the pathogenicity of a particular variant. Pathogenic variants of the GJB2 gene encoding the transmembrane protein Cx26 are one of the most common causes of hearing loss in many human populations. Previously, based on the HeLa cell line, we obtained subclonal lines with a knockout of the GJB2 gene using the CRISPR/ Cas9 system. One of them served as the basis for generating transgenic cell lines with constant expression of a number of mutant GJB2 variants identified in patients with hearing loss. The aim of this work is to characterize and further develop a panel of transgenic HeLa cell lines stably expressing various variants of the GJB2 gene for comparative functional analysis of the effects of mutant GJB2 variants on the structure and functions of the Cx26 protein. Taking into account the features of the «life cycle» of the Cx26 protein, the approaches have been defined and a set of experimental methods have been developed to assess the expression of mutant GJB2 variants, to assess the intracellular localization and ability of mutant forms of the Cx26 protein to form functional hemichannels/channels.

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