B6;129S2-Oprd1^tm2Kff/J

Stock number: 029012
Product name: DOR-EGFP
Research areas: Research Tools

Description

DOR-EGFP reporter mice have the opioid receptor, delta 1 (Oprd1) gene driving expression of an enhanced green fluorescent protein. These mice may be useful as a means for visualizing receptor neuroanatomy and real-time receptor trafficking in live neurons.

Detailed description

DOR-EGFP reporter mice have an enhanced green fluorescent protein (EGFP) knocked into the last exon of the opioid receptor, delta 1 (Oprd1) gene. DOR-EGFP mice have EGFP inserted just upstream of the stop codon of the targeted gene, allowing this strain to express endogenous Oprd1-encoded receptors with a fused EGFP at the C-terminus. Mice homozygous for this allele are viable and fertile. δ-opioid receptors (DORs) are G-protein-coupled receptors that inhibit neuronal activity upon binding of ligands such as enkephalins and other opioids. DORs are targets for the treatment of chronic pain, drug abuse, and emotional disorders.

Mice emit EGFP fluorescence in neurons in the caudate putamen at birth, in the hippocampus at day 3 after birth, with increasing expression throughout the brain reaching maximal intensity at day 15. Although, Oprd1 transcription, receptor numbers, and activation is slightly increased in DOR-EGFP mice compared to wild-type animals, affinity of the receptor for agonists and endogenous peptides is unchanged. Treatment with receptor agonists triggers receptor internalization, with cell surface fluorescence decreasing in a dose-dependent manner. This correlates with a rapid and dose-dependent increase of locomotor activity. Mice with internalized receptors are insensitive to subsequent agonist administration.

When crossed to MOR-mCherry mice (Stock No. 029013), expressing mCherry in mu opioid receptors, bicolor double mutant mice (DOR-eGFP/MORmcherry) express functional fluorescent forms of the two receptors.

Development

A targeting vector was designed by Dr. Brigitte Kieffer's reasearch team to insert an enhanced green fluorescent protein (EGFP), and a loxP-flanked hygromycin resistance (hygro) cassette, in exon 3, just upstream of the stop codon, of the opioid receptor, delta 1 (Oprd1) coding region. The construct was electroporated into 129svPas-derived embryonic stem (ES) cells. Correctly targeted ES cells were transiently transfected with a cre recombinase expression plasmid to delete the hygro cassette and resulting chimeric mice were crossed C57BL/6J mice (Stock No. 000664). Upon arrival at The Jackson Laboratory, cryopreserved sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664) to establish our live colony of DOR-EGFP reporter mice.

Allele

Symbol: Oprd1^tm2Kff
Name: targeted mutation 2, Brigitte L Kieffer
MGI accession ID: MGI:3653589
Generation method: Targeted
Attributes: Reporter; Null/Knockout
Marker symbol: Oprd1
Marker name: opioid receptor, delta 1
Chromosome: 4
Strain of origin: 129S2/SvPas
Expressed genes: GFP,Green Fluorescent Protein,
Site of expression: EGFP fluorescence is expressed in neurons of the brain.
Molecular note: Homologous recombination was used to introduce the EGFP cDNA into exon 3 in frame and upstream of the stop codon. Western blot demonstrated expression of an EGFP protein as expected. Quantitative mRNA analysis demonstrated that the insertion did not interrupt normal transcription of the locus.