The targeting vector contains an endoplasmic reticulum retention signal and an IRES-PLAP reporter gene, allowing extracellular antibody staining/chromogenic development tracking of cells normally expressing the endogenous gene. Homozygous mice display behavioral deficits in response to mustard oil, cold, and punctate mechanical stimuli. These mutants may be useful in neurobiological studies involving dorsal root ganglion neurons and cells of the inner ear, as well as for auditory, temperature, or chemical irritant trials.
David P. Corey, Harvard Medical School/HHMI
Homozygous mice are viable and fertile. The donating investigator reports a dramatic loss of fecundity after 5-6 months of age. The targeting vector contains an endoplasmic reticulum (ER) retention signal (KDEL), which is reported to sequester the potential truncated mRNA product in the ER. The vector also contains an IRES-PLAP reporter gene, allowing extracellular antibody staining/chromogenic development tracking of cells normally expressing the endogenous gene. Homozygous mice display behavioral deficits in response to mustard oil, cold, and punctate mechanical stimuli. These mice have a normal startle reflex to loud noise, a normal sense of balance, a normal auditory brainstem response, and normal transduction currents in vestibular hair cells. These mutants may be useful in neurobiological studies involving dorsal root ganglion neurons and cells of the inner ear, as well as for auditory, temperature, or chemical irritant trials.
A "KDEL-IRES-PLAP-pA-FRT-PGKNeo-FRT" targeting vector was created with an in-frame endoplasmic reticulum (ER) retention signal and a stop codon (KDEL), internal ribosome entry site (IRES)-controlled human placental alkaline phosphatase gene (PLAP) and a polyadenylation sequence all followed by a FRT-flanked PGK-Neo cassette. This construct was designed to replace the endogenous exons encoding for the pore domain (S5 and S6 transmembrane domains and the pore loop that contains the selectivity filter). The construct was electroporated into the 129P2/OlaHsd-derived E14 embryonic stem (ES) cells. Correctly targeted ES cells (with the selection cassette removed) were injected into C57BL/6 blastocysts. Chimeric males were bred to C57BL/6 or B6129P1/F2J females. Heterozygotes were bred together and maintained on a mixed B6;129 background prior to arrival at The Jackson Laboratory.
|Allele Name||targeted mutation 1, Kelvin Y Kwan|
|Allele Type||Targeted (Null/Knockout)|
|Allele Synonym(s)||Trpa1 KO|
|Gene Symbol and Name||Trpa1, transient receptor potential cation channel, subfamily A, member 1|
|Strain of Origin||129P2/OlaHsd|
|Molecular Note||The S5 and S6 transmembrane domains and the pore loop containing the selectivity filter of the gene were replaced with an IRES-PLAP-pA cassette. The resulting sequence produces a bicistronic transcript containing the 5' end of the endogenous gene followed by an independently translated human placental alkaline phosphatase gene. An endoplasmic reticulum retention signal encoded by the amino acid sequence KDEL and a stop codon were inserted in frame with the exon before the IRES-PLAP to prevent unwanted side effects from a possibly truncated product of the endogenous coding sequence. RT-PCR confirmed absence of transcript in mutants.|
|Mutations Made By|| |
Kelvin Kwan, Rutgers University
When maintaining a live colony, the donating investigator breeds heterozygous and homozygous mice together. Homozygous mice have some mild subfertility problems.
When using the Trpa1 KO mouse strain in a publication, please cite the originating article(s) and include JAX stock #006401 in your Materials and Methods section.