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A hearing and vestibular phenotyping pipeline to identify mouse mutants with hearing impairment

Nature Protocols volume 5, pages 177–190 (2010)Cite this article

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Abstract

We describe a protocol for the production of mice carrying N-ethyl-N-nitrosourea (ENU) mutations and their screening for auditory and vestibular phenotypes. In comparison with the procedures describing individual phenotyping tests, this protocol integrates a set of tests for the comprehensive determination of the causes of hearing loss. It comprises a primary screen of relatively simple auditory and vestibular tests. A variety of secondary phenotyping protocols are also described for further investigating the deaf and vestibular mutants identified in the primary screen. The screen can be applied to potentially thousands of mutant mice, produced either by ENU or other mutagenesis approaches. Primary screening protocols take no longer than a few minutes, apart from ABR testing which takes upto 3.5 h per mouse. These protocols have been applied for the identification of mouse models of human deafness and are a key component for investigating the genes and genetic pathways involved in hereditary deafness.

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Figure 1: Breeding schemes for generating both G1 and G3 pedigrees from N-ethyl-N-nitrosourea (ENU) mutagenized males.
Figure 2: The hearing and vestibular phenotyping pipeline as carried out at MRC Harwell.
Figure 3: Primary hearing and vestibular testing of mice.
Figure 4: Auditory brainstem response (ABR) testing.
Figure 5: Dissections of mouse ossicles.
Figure 6: Dissection of the inner ear for scanning electron microscopy (SEM).
Figure 7: Parasaggital hematoxylin and eosin (H&E) sections through the saccular maculae of an otoconial mutant mouse and a wild-type mouse.
Figure 8: Scanning electron micrographs showing the sensory hair cells on the organ of Corti of a wild-type mouse.
Figure 9: Middle ear ossicles from P20 Pdo/+ mutants (a) and controls (b) showing malformed articulatory surfaces in all three bones.
Figure 10: Histopathology of the middle ear.
Figure 11: Auditory brainstem response (ABR) analysis of the Cloth-ears mutant.

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Acknowledgements

This work was supported by the MRC and FP6 Integrated Project EUROHEAR, LSHG-CT-2004-512063. We thank Steve Thomas and Kevin Glover for figure preparation.

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Authors and Affiliations

  1. MRC Harwell, Mammalian Genetics Unit, Harwell Science and Innovation Campus, Harwell, UK

    Rachel E Hardisty-Hughes, Andrew Parker & Steve D M Brown

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  1. Rachel E Hardisty-Hughes
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Contributions

R.E.H.-H. and S.D.M.B. designed the pipeline; R.E.H.-H. and A.P. carried out experimental work utilizing the pipeline; and R.E.H.-H., A.P. and S.D.M.B. wrote the paper.

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Correspondence to Steve D M Brown.

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Hardisty-Hughes, R., Parker, A. & Brown, S. A hearing and vestibular phenotyping pipeline to identify mouse mutants with hearing impairment. Nat Protoc 5, 177–190 (2010). https://doi.org/10.1038/nprot.2009.204

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