Journal of Otolaryngology and Ophthalmology of Shandong University ›› 2026, Vol. 40 ›› Issue (1): 6-12.doi: 10.6040/j.issn.1673-3770.0.2024.667

• Original Article • Previous Articles     Next Articles

In vivo proximity labeling of cell-surface proteins in mouse cochlear hair cells

WANG Nuan1,2, WANG Xinyang1,2, JI Changhao1,2, LIU Min2, GAO Jiangang2,3, SUN Jin1,2   

  1. 1. School of Laboratory Animal, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan 250117, Shandong, China2. Medical Science and Technology Innovation Center, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan 250117, Shandong, China3. School of Life Science, Key Laboratory of the Ministry of Education for Experimental Teratology, Shandong University, Jinan, 250100, Shandong, China
  • Online:2026-01-20 Published:2026-02-13

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Abstract: Objective To develop a method for labeling cell-surface proteins of cochlear hair cells in mice and establish conditions for profiling the cell-surface proteome of these cells. Methods A recombinant adeno-associated virus(rAAV)was engineered to express a horseradish peroxidase fusion protein(HRP-TM)specifically in hair cells, targeting the outer surface of the cell membrane. The virus was microinjected into the posterior semicircular canal of 2-day-old C57BL/6 mice. At 1 month of age, auditory brainstem response(ABR)was measured, and HRP expression and protein labeling were assessed via basilar membrane immunofluorescence staining. Results ABR results showed no significant hearing differences between virus-injected and control mice. Immunofluorescence revealed HRP-TM was specifically expressed in hair cells, with >95% transduction efficiency in inner hair cells and ~80% transduction efficiency in outer hair cells. Hair cell morphology and arrangement remained normal. Fluorescent streptavidin staining confirmed membrane-specific labeling exclusively in HRP-TM-expressing hair cells. Conclusion The rAAV-HRP-TM system efficiently and specifically labels cochlear hair cell surface proteins without cytotoxicity or functional impairment, providing a foundation for isolating and analyzing these proteins via mass spectrometry.

Key words: Cochlear hair cells, Proximity labeling, rAAV, HRP, Cell-surface proteome

CLC Number: 

  • Q95-3
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