Abstract
The otocyst, an anlage of the inner ear, presents an attractive target to study treatment strategies for genetic hearing loss and inner ear development. We have previously reported that electroporation-mediated transuterine gene transfer of Connexin30, utilizing a monophasic pulse into Connexin30−/− mouse otocysts at embryonic day 11.5, is able to prevent putative hearing deterioration. However, it is not clear whether supplementary gene transfer can rescue significant morphological changes, caused by genetic deficits. In addition, with the transuterine gene transfer technique utilized in our previous report, the survival rate of embryos and their mothers after treatment was low, which became a serious obstacle for effective in vivo experiments. Here, we set out to elucidate the feasibility of supplementation therapy in Slc26a4 deficient mice, utilizing biphasic pulses, optimized by modifying pulse conditions. Modification of the biphasic pulse conditions during electroporation increased the survival rate. In addition, supplementation of the target gene cDNA into the otocysts of homozygous Slc24a4 knockout mice significantly prevented enlargement of the endolymphatic space in the inner ear areas; moreover, it rescued hearing and vestibular function of mice in vivo.
Highlights
The otocyst, an anlage of the inner ear, presents an attractive target to study treatment strategies for genetic hearing loss and inner ear development
To determine the optimized pulse condition regarding both survival and expression rates, EGFP expression was assessed at E 13.5 by fluorescent microscope after treatment with monophasic (M) (Fig. 1A) and biphasic (B) pulses (Fig. 1B) at E 11.5, respectively (Supplemental Table 1)
Survival rate improved when the total energy was reduced, but this resulted in a decrease of the EGFP expression (Fig. 1C and Supplemental Table 2B)
Summary
The otocyst, an anlage of the inner ear, presents an attractive target to study treatment strategies for genetic hearing loss and inner ear development. We have previously reported that electroporationmediated transuterine gene transfer of Connexin[30], utilizing a monophasic pulse into Connexin30−/− mouse otocysts at embryonic day 11.5, is able to prevent putative hearing deterioration. It is not clear whether supplementary gene transfer can rescue significant morphological changes, caused by genetic deficits. Supplementation of the target gene cDNA into the otocysts of homozygous Slc24a4 knockout mice significantly prevented enlargement of the endolymphatic space in the inner ear areas; it rescued hearing and vestibular function of mice in vivo. Slc26a4 deleted mice display an enlargement of the endolymphatic space followed by a failure to develop normal hearing and balance[25,26]
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