STOCK Trpm8^tm1Apat/J

Stock number: 014581
Research areas: Neurobiology Research, Research Tools, Sensorineural Research

Description

These TRPM8^EGFPf targeted mutation (knock in) mice display deficits in cool thermosensation and may be useful in studies of cold nociception.

Detailed description

These mutant mice express farnesylated enhanced GFP gene (EGFPf) from the endogenous Trpm8 locus. Mice that are homozygous for the targeted mutation are viable, fertile, normal in size and do not display any gross physical or behavioral abnormalities. No wildtype gene product (mRNA) is detected although low levels of a truncated mRNA splice variant is sometimes detected by RT-PCR analysis of total RNA derived from dorsal root ganglia (DRG) isolated from homozygotes. No Trpm8 locus mRNA is detected by in situ hybridization of DRG isolated from homozygotes. In thermotaxis assays performed in a cage equipped with surface temperature gradients, homozygous mice spend less time in the warmer area compared to wildtype controls. Homozygotes display diminished responses to application of acetone and injection of icilin. Noxious cold sensation of colder temperatures (sub zero) is normal. Mutant mice do not display cold-induced analgesia in response to pain. EGFPf-positive neurons isolated from homozygotes are more sensitive to cold and menthol. TRPM8-expressing neurons isolated from mutants are specifically labeled by EGFPf expression. EGFPf fluorescence is detected in the dorsal root ganglia and spinal cord from both homozygotes and heterozygotes (with an increased fluorescence intensity observed in homozygotes). Axonal terminals are visualized utilizing immunohistochemical methods.

Development

A targeting vector containing a farnesylated enhanced GFP gene (EGFP-F), SV40polyA signal sequence, and a loxP site flanked EM7-PGK-Neo cassette, was used to replace sequence encoding amino acids 2-29. The construct was electroporated into 129S1/Sv-Oca2⁺ Tyr⁺ Kitl⁺ derived CJ7 embryonic stem (ES) cells. Correctly targeted ES cells were injected into C57BL/6 blastocysts. The resulting male chimeric animals were crossed to C57BL/6 female mice. The mice were crossed to an EIIA-Cre deleter strain (Stock No. 003724) to remove the floxed Neo cassette. Upon arrival at The Jackson Laboratory, the mice were crossed to C57BL/6J (Stock No. 000664) at least once to establish the colony.

Allele

Symbol: Trpm8^tm1Apat
Name: targeted mutation 1, Ardem Patapoutian
MGI accession ID: MGI:3716558
Generation method: Targeted
Attributes: Reporter; Null/Knockout
Marker symbol: Trpm8
Marker name: transient receptor potential cation channel, subfamily M, member 8
Chromosome: 1
Strain of origin: 129S1/Sv-Oca2⁺ Tyr⁺ Kitl⁺
Expressed genes: GFP,Green Fluorescent Protein,
Site of expression: Farnesylated EGFP (EGFPf) fluorescence is detected in the dorsal root ganglia and spinal cord.
Molecular note: Velocigene technology was used to delete sequences encoding amino acid residues 2-29 and insert a farnesylated enhanced GFP gene followed by an SV40polyA tail in frame with the start codon of the endogenous gene. RT-PCR confirmed absence of wild-type transcript in mutants, however, in some samples a truncated splice variant was detected at low levels.