Dottorato in BIOLOGIA CELLULARE E DELLO SVILUPPO

Titolo della tesi: Mutations of Cx26 gene affecting inner ear and skin: Characterization of two mutant mouse models.

The GJB2 gene encodes protein called gap junction beta 2, also known as connexin 26 (Cx26), a member of the connexin protein family which form intercellular gap junction channels that permit the transport of nutrients, charged atoms (calcium and potassium ions), and signaling molecules between adjoining cells. Cx26 is found in cells throughout the body, including the inner ear and the skin, and plays an important role in hearing as well as growth, maturation, and stability of the epidermis. In this study, we focused on two Cx26 point mutations, V84L and D50N, respectively associated with non-syndromic hearing loss and deafness (DFNB1) and keratitis-ichthyosis-deafness (KID) syndrome. We produced two mutant mice and phenotyped them. To phenotype Cx26V84L mutant mice, obtained with the Crispr/Cas9 technique, we used Auditory Brainstem Response (ABR) recordings to quantify the degree of hearing loss. To phenotype Cx26D50N mice, we used Micro Computed Tomography (Micro-CT) analysis. Since the D50N mutation is dominant and embryonically lethal, we used a classical technique to produce mutant mice, i.e. blastocyst injection of embryonic stem cells using a LoxP-stop-LoxP construct (or “LSL cassette”). The latter presents a stop codon within LoxP sites on either side upstream of the Cx26D50N gene that prevents gene expression in the absence of Cre. The first mutant, Cx26v84L, was a conditional Cre-inducible mouse model and we mated it with two different Cre-strains: (1) CreERT2 homozygous reporter mice for ubiquitous transgene expression and (2) Sox10-CRE deleter mice, for a constitutive expression of the mutated gene at the ear level. As for the first mating, there was no expression of mutant transcript after Tamoxifen treatment, because the organ of Corti develops during embryogenesis and the induction with the drug, in the adult, was not enough to yield a strong induction of the mutated gene. This result was confirmed by ABR recordings, which showed mice had normal hearing. In contrast, mating with Sox10-CRE deleter mice resulted in the expression of the V84L point mutation, and mice with hearing loss. The second model, Cx26D50N, was a Knock-in mouse with some phenotypical characteristics of KID syndrome. In particular, all mutant pups were trans-lucid, had tail necrosis, developmental delay of the limbs and growth retardation. Eventually, they died soon after birth.