B6.129(SJL)-Cnr1^tm1.1Kpm/J

Stock number: 026849
Product name: S426A/S430A mutant mice
Research areas: Neurobiology Research, Research Tools

Description

These S426A/S430A mutant mice exhibit increased sensitivity to cannabinoids, delayed development of tolerance to THC and enhanced cannabinoid dependence. The may be suitable for use in studies of cannabinoid receptor 1-mediated effects on metabolism, drug addiction and learning.

Detailed description

The Cnr1 gene encodes the cannabinoid receptor 1, which is involved in cannabinoid-induced central nervous system effects by inhibiting adenylate cyclase activity in a dose-dependent, pertussis toxin-sensitive manner. Cannabinoid receptor 1, like other G protein coupled receptors, can be desensitized by continuous activation, through phosphorylation by GPCR kinases (GRKs) and subsequent ßarrestin binding. Chronic administration of cannabinoids leads to the rapid development of tolerance. These mice express a desensitization resistant form of cannabinoid receptor 1, with an N-terminal HA tag. The two point mutations that change serines 426 and 430 to alanines, S426A and S430A, are putative GRK3 phosphorylation sites. The mutant receptor was detected by PCR analysis. Western blot analysis confirmed that protein levels in the brain remain unchanged except for a 15% decrease in the cerebellum. Ligand affinity and G-protein activation were not altered in the mutant receptor. Homozygous mice exhibit increased sensitivity to endogenous and exogenous cannabinoids, delayed development of tolerance to THC (delta-9-tetrahydrocannabinol), and enhanced cannabinoid dependence (increased severity of withdrawal symptoms). Mice that are homozygous for the targeted mutation are viable and fertile.

Development

A targeting vector containing an FRT site flanked NEO cassette was used to insert 2 point mutations, S426A and S430A (putative GRK phosphorylation sites), and a N-terminal HA tag into the targeted gene. The construct was electroporated into 129 derived R1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts. The resulting chimeric animals were tested for germline transmission. The mice were then bred to transgenic mice expressing FLPe recombinase (Stock No. 005703) to remove the FRT flanked selection cassette. The mice were then backcrossed to C57BL/6J for approximately 15 generations. 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: Cnr1^tm1.1Kpm
Name: targeted mutation 1.1, Ken Mackie
MGI accession ID: MGI:5607776
Generation method: Targeted
Attributes: Not Specified
Synonyms: S426A/S430A
Marker symbol: Cnr1
Marker name: cannabinoid receptor 1 (brain)
Marker synonyms: CANN6; CB1; CB1A; CB1K5; CB1R; CB-R; CNR; SKR6R
Chromosome: 4
Strain of origin: (129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Expressed genes: HA tag,HA tag,
Site of expression: Central nervous system
Molecular note: An HA epitope sequence, two mutations that change serines 426 and 430 to alanines (S426A, S430A), and a neomycin phosphotransferase (NeoR) gene flanked by frt sequences were inserted via homologous recombination. Serines 426 and 430 are putative GRK phosphorylation sites that when mutated to alanines are nonphosphorylatable and result in a desensitization resistant form of Cnr1. The frt-flanked NeoR gene was removed via flp-mediated recombination. Southern blot confirmed expression of a 4.2 kb mutant fragment and PCR confirmed expression of the mutant mRNA. Western blot analysis confirmed that protein levels in the brain remain unchanged except for a 15% decrease in the cerebellum.