B6.Cg-Pou4f3^{tm1.1(HBEGF)Jsto}/RubelJ

Stock number: 028673
Product name: Pou4f3^DTR
Research areas: Research Tools, Sensorineural Research

Description

The coding region of human diptheria toxin receptor (HBEGF) was introduced upstream of the translation initiation site of the mouse Pou4f3 (POU domain, class 4, transcription factor 3) gene. Hair cells can be specifically depleted from the cochlea and vestibule with diphtheria toxin treatment. These mice may be suitable for use in studies related to development and regeneration in the auditory and vestibular systems.

Detailed description

The POU domain, class 4, transcription factor 3, encoded by the Pou4f3 gene, is expressed in auditory and in vestibular hair cells, retina, striatum and brain stem. Mutations in this gene are associated with progressive hearing loss and deafness. These Pou4f3^DTR knock-in mice express the human diptheria toxin receptor (HBEGF) from the endogenous Pou4f3 locus. Heterozygous Pou4f3^DTR/+ mice have a normal complement of inner ear hair cells and normal hearing and balance. Diphtheria toxin treatment on cochlear explants from neonatal heterozygous mice ablates inner hair cells completely and outer hair cells almost completely. In mature heterozygous mice, by 3 days postinjection of diphtheria toxin (25ng/g dose), auditory brainstem responses (ABRs) indicate profound hearing loss and by 5 days post-injection the mice are totally deaf. No hearing recovery is observed, at least out to 4 months post-injection. A smaller diphtheria toxin dose of 10 ng/g delays the hearing loss by one or 2 days. At 6 days postinjection (25 ng/g), almost all hair cells are ablated, with loss starting in the basal turn proceeding to the apex. Inner hair cells are ablated more slowly than outer hair cells. In mature mice, treatment does not result in neuronal loss. In young heterozygous mice, postnatal day 5, a dose of 5 ng/g diphtheria toxin results in loss of hair cells before hearing onset. In neonates, most hair cells (<1% remaining) are ablated by 8 days post-injection with transneuronal loss of neurons in the ventral cochlear nucleus and spiral ganglion neuron. Supporting cells are not altered by diphtheria toxin exposure. Mice that are heterozygous for the targeted mutation are viable and fertile. Homozygotes (untreated) exhibit severe ataxia, vestibular defects and have not been tested for fertility.

Development

A targeting vector containing the full coding region of human diphtheria toxin receptor (HBEGF) gene and a floxed SvNeo gene was inserted upstream of the Pou4f3 translation initiation site, into exon 1. The construct was electroporated into unspecified B6C3 derived embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts. The resulting chimeric animals were tested for germline transmission. The mice were crossed to Mox2-Cre mice, on the C57BL/6 background, to excise the loxP site flanked SvNeo gene. The mice were then backcrossed to C57BL/6 for 10 generations, subsequently removing the Mox2-Cre allele. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

Allele

Symbol: Pou4f3^{tm1.1(HBEGF)Jsto}
Name: targeted mutation 1.1, Jennifer S Stone
MGI accession ID: MGI:5467563
Generation method: Targeted
Marker symbol: Pou4f3
Marker name: POU domain, class 4, transcription factor 3
Chromosome: 18
Strain of origin: C57BL/6 x C3H
Molecular note: The full coding region of human diphtheria toxin receptor (HBEGF) and a floxed neo cassette were inserted upstream of the translation initiation site. Cre-mediated recombination removed the neo cassette.