The Role of Hair Bundle Protein 7(HBP7) in the Auditory Hair Cell of Zebrafish in Mediating Hearing Function

Overview

Hair cells in the inner ear detect sounds through deflection of their stereocilia, which activate transduction channels in vertebrates, including zebrafish. It is hypothesized, from protein sequence similarity and expression profiling using RNA in situ hybridization, that HBP7 (hair bundle protein 7) is necessary for hair bundle function of inner ear hair cells. To test HBP7’s role in hearing using mutagenesis, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) mediated gene editing was used to generate a loss-of-function mutation. First, an enzyme, Cas9, cuts the two strands of target DNA at a specific gene location guided by a pre-designed RNA: single guide RNA (SgRNA). Cas9 was injected into zebrafish embryos for sgRNA to activate the Cas9 endonuclease for HBP7 mutation. Then, it was confirmed if HBP7 was mutated. Genomic DNA was extracted from the embryos and amplified using PCR. PCR products were sequenced to confirm the mutation. Specifically, the PCR products were transferred onto host bacteria through vectors and the hosts incubated. The HBP7 gene amplicons were then extracted and sequenced. The first step of confirming the hypothesis was completed when it was established that the CRISPR method successfully mutated HBP7 by comparing the sequence of the wild-type HBP7 gene to the gene fragment in the vector. Next, hearing will be examined in the homozygous HBP7 mutant population using FM1-43 dye uptake method and microphonic potential recordings that determine the value of HBP7 to sound perception. Recordings detect electrical responses to mechanical stimuli. If HBP7 is important in hair cell function, then HBP7 mutant zebrafish would have a different response to stimuli than wild-type fish.

Competition history

• AJAS 2018 Category not listed

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