Creation And Identification Of DFNA64 Mouse Models

A new DFNA locus named DFNA64, was mapped in an autosomal dominant non-syndromic hearing loss family. By the means of direct sequencing of candidate genes in mapped region, heterozygous missense mutation c.377C>T was identified in DIABLO gene, which was segregated with the disease phenotype. DIABLO plays a key role in the promotion of apoptosis by binding the inhibitor of apoptosis protein. The previous investigations suggest mutant DIABLO while retaining the pro-apoptotic function, triggers significant degradation of both wildtype and mutant Smac/Diablo, rendering host mitochondria susceptible for calcium-induced membrane potential loss.. Mutant mouse models are necessary to understand the mechanism of inner ear hair cells damage by mutant DIABLO.1. Creation and identification of DFNA64 transgenic mouse modelIn this study, we obtained two lines of transgenic mouse models expressing wild and mutant diablo respectively by microinjection of linearized plasmid construct into fertilized eggs of CD1 strain mice. The construct is derived from pcDNA3.1(+), which is designed for high-level stable and transient expression in mammalian hosts. It contains CMV promotor, mutant or wild cDNA fragment of diablo, artifical intron and bGH-polyA signal. After identified the genotype of founders, generate transgenic mouse by cross the founders. Real-time PCR identified the overexpression of both wild and mutant diablo in nervous system.2.Creation of DFNA64 knock-in mouse modelWe also established knock-in mouse model by gene targeting. Targeting ES cells from 129 strain mice by electro-transformation, with construct consisting of two homologous sequences with floxed neo castle and mutation inserted. The targeted ES cells were screened out by antibiotic G418 and PCR reaction, then microinjected into embryos. We obtained five chimera mice. DFNA64 mouse models play significant roles in the study into gene function of DIABLO, mechanism of hearing loss, prevention before onset, therapeutic approaches.