B6.Cg-Tacr3^{tm1.1(cre/GFP)Rpa}/J

Stock number: 035576
Product name: Tacr3^iCre:GFP
Research areas: Research Tools

Description

Tacr3^iCre:GFP knock-in mice should express iCre:GFP wherever Tacr3 (tachykinin receptor 3), a G protein-coupled receptor (GPCR), is normally expressed. Mice demonstrate no overt phenotype. Expression levels of the targeted gene have not been assessed, but no effect is anticipated as the insertion is beyond the termination codon.

Detailed description

Tacr3^iCre:GFP knock-in mice should express iCre:GFP wherever Tacr3 (tachykinin receptor 3), a G protein-coupled receptor (GPCR), is normally expressed. Mice demonstrate no overt phenotype. Expression levels of the targeted gene have not been assessed, but no effect is anticipated as the insertion is beyond the termination codon.

Tacr3 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.

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 IRES-mnCre:GFP was inserted just 3' of the termination codon in the last coding exon of the Tacr3 gene. The 5' arm (~4 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 Tacr3 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 IRES-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 IRES-mnCre:GFP cassette has an internal ribosome entry sequence (IRES), 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 iCre: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 EcoRV using a ³²P-labeled probe downstream of the 3' arm of the targeting construct. Five of 77 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.

Allele

Symbol: Tacr3^{tm1.1(cre/GFP)Rpa}
Name: targeted mutation 1.1, Richard D Palmiter
MGI accession ID: MGI:6451481
Generation method: Targeted
Attributes: Recombinase-expressing; Reporter
Marker symbol: Tacr3
Marker name: tachykinin receptor 3
Marker synonyms: HH11; neuromedin K receptor; NK3; NK3R; NK-3R; Nkr; Nmkr; TAC3R; TAC3RL
Chromosome: 3
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Molecular note: A cassette encoding IRES-mnCre:GFP (an internal ribosome entry sequence (IRES), 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 3' of the termination codon in the last coding exon of the Tacr3 gene via homologous recombination. The neomycin cassette was excised through crosses with animals broadly expressing Flp recombinase.