B6.Cg-Oprm1^{tm1.1(cre/GFP)Rpa}/J

Stock number: 035574
Product name: Oprm1^Cre:GFP KIKO
Research areas: Neurobiology Research, Research Tools

Description

Oprm1^Cre:GFP knock-in/knock-out mice express Cre:GFP wherever Oprm1 (opioid receptor, mu 1), a G protein-coupled receptor (GPCR), is normally expressed. Heterozygotes demonstrate no overt phenotype. Homozygotes lack mu opioid receptor expression and hence no longer respond to certain endogenous or exogenous ligands. These mice allow cre recombinase-dependent expression of effector/reporter genes in cells that normally express this receptor.

Detailed description

Oprm1^Cre:GFP knock-in/knock-out mice express Cre:GFP wherever Oprm1 (opioid receptor, mu 1), a G protein-coupled receptor (GPCR), is normally expressed. Heterozygotes demonstrate no overt phenotype. Homozygotes lack mu opioid receptor expression and hence no longer respond to certain endogenous or exogenous ligands. These mice allow cre recombinase-dependent expression of effector/reporter genes in cells that normally express this receptor.

Oprm1 is normally not expressed abundantly, thus the GFP fluorescence is not directly visible and barely detectable with antibodies to GFP. More sensitive RNAscope in situ technology may better reveal expression patterns, however. Update February, 2022: We have received a report that endogenous GFP fluorescence is more than sufficient to be visualized by confocal microscopy in at least some regions of the brain (e.g. the medial habenula and lateral habenula).

The donating laboratory never attempted to visualize expression of a Myc tag component that is part of the knock-in construct. Since antibody to GFP does not work well in this strain, it is suspected that antibody to the Myc tag will not either.

This strain has been tested with adeno-associated virus expressing double-floxed inverse ORF fluorescent protein (AAV‐DIO‐FP; either GFP or mCherry) injected into a brain region where expression was expected and it revealed robust fluorescence.

Development

A cassette encoding Cre:GFP was inserted just 5' of the initiation codon in the first coding exon of the Oprm1 gene. The 5' arm (~3.1 kb with PacI and SalI sites at 5' and 3' ends, respectively) and 3' arm (~4.5 kb with XhoI and NotI sites at 5' and 3' ends, respectively) of the Oprm1 gene were amplified from a C57BL/6 BAC clone by PCR using Q5 Polymerase (New England Biolabs) and cloned into polylinkers of a targeting construct that contained mnCre:GFP, a FRT-flanked Sv40Neo gene for positive selection, and HSV thymidine kinase and Pgk-diphtheria toxin A chain genes for negative selection. The mnCre:GFP cassette has a Myc-tag and nuclear localization signals at the N-terminus of Cre recombinase, which is fused to green fluorescent protein followed by a SV40 polyadenylation sequence (abbreviated here as Cre:GFP).

The construct was electroporated into (129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem (ES) cells. Correct targeting was determined by Southern blot of DNA digested with Kpn1 using a ³²P-labeled probe downstream of the 3' arm of the targeting construct. Twelve of 84 clones analyzed were correctly targeted. One clone that was injected into blastocysts resulted in good chimeras that transmitted the targeted allele through the germline. Progeny were bred with Gt(Rosa)26Sor-FLP recombinase mice to remove the FRT-flanked SV-Neo gene. Mice were then backcrossed to C57BL/6J mice for more than 6 generations by the donating laboratory.

In 2020, a 48 SNP (single nucleotide polymorphism) panel analysis, with 43 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the males sent to The Jackson Laboratory Repository. Two of the 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6N;C57BL/6J genetic background.

Allele

Symbol: Oprm1^{tm1.1(cre/GFP)Rpa}
Name: targeted mutation 1.1, Richard D Palmiter
MGI accession ID: MGI:6451472
Generation method: Targeted
Attributes: Recombinase-expressing; Reporter; Null/Knockout
Synonyms: Oprm1^Cre:GFP; Oprm1^Cre
Marker symbol: Oprm1
Marker name: opioid receptor, mu 1
Marker synonyms: LMOR; MOP; MOP receptor; MOP-R; mor; MOR1; M-OR-1; MOR-1; MORA; muOR; Oprm; Oprrm1
Chromosome: 10
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Molecular note: A cassette encoding Cre:GFP (a Myc-tag and nuclear localization signals at the N-terminus of Cre recombinase, which is fused to green fluorescent protein followed by a SV40 polyadenylation sequence) was inserted just 5' of the initiation codon in the first coding exon of the Oprm1 gene replacing the exon 1 via homologous recombination. The neomycin cassette was excised of through crosses with animals broadly expressing Flp recombinase.