431 Evaluating defective Transcription Coupled-Nucleotide Excision Repair as a mechanism for sensorineural hearing loss in a zebrafish model of Cockayne Syndrome

Abstract

OBJECTIVES/GOALS: The characterization of the zebrafish as an animal model for Cockayne Syndrome may guide us towards role of Transcription-Coupled Nucleotide Excision Repair (TC-NER) defects in sensorineural hearing loss. METHODS/STUDY POPULATION: To examine our model, we have developed a zebrafish line with a 9+1 base-pair deletion in the ercc6 gene using TALENs. Mutation has since been confirmed by PCR and subsequent restriction digest with StuI. A series of assays evaluating hair cell morphology, structure and function, as well as ribbon synapse structure, will be used to analyze potential differences between the ercc6 mutant zebrafish line a their wild-type siblings. Additionally, electron microscopy will be used to assess differences in hair cell ultrastructure between the ercc6 mutant zebrafish line a their wild-type siblings. Finally, UVC exposure assays will be used to determine the role TC-NER plays in our novel zebrafish model, and evaluate its potential implications in sensorineural hearing loss. RESULTS/ANTICIPATED RESULTS: We anticipate that biallelic loss of function mutations in the zebrafish ercc6 gene will result in abnormalities in hair cell structure, mechanotransduction, or cell number. Additionally, we anticipate that hair cell ultrastructure and ribbon synapse structure will be impacted by loss of ercc6 expression. DISCUSSION/SIGNIFICANCE: Hearing loss mechanisms associated with defects in TC-NER are yet to be described. We believe our model will provide the tools for a faster and efficient way to carry out Cockayne Syndrome studies while laying the groundwork for the association between TC-NER and hearing loss.