Abstract
The inner ear consists of the cochlea (the organ of hearing) and the vestibular system (the organs of balance). Within the vestibular system, linear acceleration and gravity are detected by the saccule and utricle. Resting above the neurosensory epithelia of these organs are otoconia, minute proteinaceous and crystalline (calcite) inertial masses that shift under the physical forces imparted by linear movements and gravity. It is the transduction and sensation of these movements and their integration with vision and proprioceptive inputs that contribute to the sensation of balance. It has been proposed that a reactive oxygen species- (ROS-) generating NADPH oxidase comprising the gene products of the Nox3, Noxo1, and Cyba genes plays a critical and constructive role in the process of inner-ear development, specifically, the deposition of otoconia. Inactivation in mouse of any of the NADPH oxidase components encoded by the Nox3, Noxo1, or Cyba gene results in the complete congenital absence of otoconia and profound vestibular dysfunction. Here we describe our use of PCR, reverse transcription-PCR (RT-PCR), and rapid amplification of cDNA ends (RACE) with traditional and high-throughput (HTP) sequencing technologies to extend and complete the molecular characterization of an allelic series of seven mutations in the Nox3 gene. Collectively, the mutation spectrum includes an endogenous retrovirus insertion, two missense mutations, a splice donor mutation, a splice acceptor mutation, premature translational termination, and a small duplication. Together, these alleles provide tools to investigate the mechanisms of otoconial deposition over development, throughout aging, and in various disease states.
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Acknowledgments
The authors acknowledge the support of the Mouse Mutant Resource (NIH/NCRR grant RR001183) and the Computational Sciences Statistics and Analysis Group, Allele Typing & Sequencing Service, Fine Mapping Laboratory, DNA Resource, and Cryopreservation Service of The Jackson Laboratory. The authors thank Ms. Leslie Haynes and The Jackson Laboratory Research Animal Facility staff for animal husbandry. The authors especially thank Dr. Yueming Ding for analysis of HTP sequence and Dr. Leah Rae Donahue, Director of the Genetic Resource Science group, for facilities and support. This work was supported by NIH/NIDCD grant DC007431 to DEB and NIH/NCI CORE grant CA34196 to JAX.
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Flaherty, J.P., Fairfield, H.E., Spruce, C.A. et al. Molecular characterization of an allelic series of mutations in the mouse Nox3 gene. Mamm Genome 22, 156–169 (2011). https://doi.org/10.1007/s00335-010-9309-z
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DOI: https://doi.org/10.1007/s00335-010-9309-z