B6.Cg-Cdh23^Ahl+ Tectb^em1Guru/GprIndzJ

Stock number: 041413
Product name: Tectb^C225Y
Research areas: Mouse/Human Gene Homologs, Neurobiology Research, Sensorineural Research

Description

Tectb^C225Y knock-in mice have a cysteine to tyrosine mutation at codon 225 of the Tectb gene that corresponds to the human variant found in autosomal dominant, non-syndromic hearing loss.

Detailed description

Tectb^C225Y knock-in mice have a cysteine to tyrosine mutation at codon 225 of the tectorin beta (Tectb) gene. Tectb encodes a non-collagenous glycoprotein component of the tectorial membrane (TM), a specialized, collagen-rich, acellular matrix of the inner ear, essential for stimulating mechanosensitive hair cell bundles during sound transduction. The structural integrity of TECTB is critical for frequency tuning and auditory sensitivity. The Tectb^C225Y mutation corresponds to the human variant found in autosomal dominant, non-syndromic hearing loss.

Mice homozygous or heterozygous for the Tectb^C225Y knock-in allele are viable and fertile. These mice are maintained with the wildtype Cdh23^Ahl allele (see Stock No. 002756), correcting the C57BL/6-susceptible early-onset age-related hearing loss phenotype. The donating investigator reports that mice homozygous for the Tectb-C225Y mutation (Tectb^C225Y/C225Y) and homozygous for the Ahl mutation (Cdh23^Ahl/Ahl) develop progressive severe hearing loss and profound disruption of TM morphology. Mice heterozygous for the Tectb-C225Y mutation (Tectb^C225Y/+) and homozygous for the Ahl mutation (Cdh23^Ahl/Ahl) display decreased matrix content within the TM and increased susceptibility to noise-induced hearing loss despite normal auditory thresholds prior to noise exposure.

Development

A guide RNA (TGTCTCATCGGTGGGGC/AGCTGG) in antisense orientation was used to create a TGC to TAC mutation in codon 225 of the tectorin beta (Tectb) gene resulting in a cysteine to tyrosine mutation (C225Y) that corresponds to the human variant found in autosomal dominant, non-syndromic hearing loss based on Tectb-201 ENSMUST00000025936.12. sgRNA, the Cas9 mRNA, and a repair-ssDNA were introduced into C57BL/6N-derived embryos via pronuclear injection. Injected embryos were transferred to pseudopregnant females and the liveborn offsprings were genotyped by PCR and sequencing and correctly targeted offspring were further bred to C57BL/6N mice for one generation. F1 offspring were bred to B6.CAST-Cdh23^Ahl+ mice (Stock No. 002756) to introduce the wildtype Cdh23^Ahl+ allele, correcting the C57BL/6-susceptible early-onset age-related hearing loss phenotype. Mice have been maintained as heterozygous or homozygous for the Tectb-C225Y mutation (Tectb^C225Y/+ or Tectb^C225Y/C225Y) and homozygous for the Ahl mutation (Cdh23^Ahl/Ahl) for at least 9 generations. It is unknown if any off-target CRISPR sites are present. Upon arrival at The Jackson Laboratory, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6NJ oocytes (Stock No. 005304).

Allele

Symbol: Tectb^em1Guru
Name: endonuclease-mediated mutation 1, Channabasavaiah Gurumurthy
MGI accession ID: MGI:8277171
Generation method: Endonuclease-mediated
Attributes: Humanized sequence
Synonyms: Tectb-C225Y
Marker symbol: Tectb
Marker name: tectorin beta
Marker synonyms: [b]-tectorin; Tctnb
Chromosome: 19
Strain of origin: C57BL/6N
Molecular note: A guide RNA (TGTCTCATCGGTGGGGC/AGCTGG) in antisense orientation was used to create a TGC to TAC mutation in codon 225 of the tectorin beta (Tectb) gene, resulting in a cysteine to tyrosine mutation (C225Y) that corresponds to the human variant found in autosomal dominant, non-syndromic hearing loss based on Tectb-201 ENSMUST00000025936.12.

Allele

Symbol: Cdh23^Ahl+
Name: wild-type of age related hearing loss
MGI accession ID: MGI:2665714
Generation method: Not Applicable
Synonyms: Cdh23^753G; Cdh23^CAST; Cdh23^{mdfw-ahl-CAST/Ei}
Marker symbol: Cdh23
Marker name: cadherin 23 (otocadherin)
Marker synonyms: 4930542A03Rik; ahl; bob; CDHR23; mdfw; nmf112; nmf181; nmf252; PITA5; sals; USH1D
Chromosome: 10
Strain of origin: CAST/EiJ
Molecular note: This variant of the SNP rs257098870 allele contrasts with the variant of the allele found in C57BL/6J in that a G is present at coding nucleotide position 753 rather than an A. Thus, the G-GT splice donor site is maintained.