B6;C3-Oxtr^{em4(icre/ERT2)Yinn}/2J

Stock number: 038989
Product name: Oxtr^{3xHA-iCreERT2} line 8
Research areas: Cancer Research, Neurobiology Research, Research Tools

Description

Oxtr^{3xHA-iCreERT2} knock-in mice express a 3xHA-tag and icre/ERT2 under direction of the endogenous oxytocin receptor (Oxtr) promoter in brain regions. This mouse strain could be useful for oxytocin receptor visualization and manipulation.

Detailed description

Oxtr^{3xHA-iCreERT2} knock-in mice express icre/ERT2 under direction of the endogenous oxytocin receptor (Oxtr) promoter. Inserted just upstream of the Oxtr stop codon, this insertion does not disrupt Oxtr endogenous transcriptional regulation. This line harbors a 3xHA-tag at the C-terminus of the receptor protein. Oxtr encodes a G-protein coupled receptor for the neuropeptide oxytocin (OXT), which is produced in the hypothalamus. OXT plays important roles in modulating social behaviors such as social recognition, social learning, pair bonding, anxiety, autism, and maternal behaviors.

When Oxtr^{3xHA-iCreERT2} mice are bred with mice containing loxP-flanked sequences, tamoxifen-inducible, cre-mediated recombination will result in deletion of the floxed sequences in the cre-expressing cells of the offspring. Homozygous mice are viable and fertile.

When bred with Ai9 cre reporter mice (Stock No. 007909), oral tamoxifen induction in double heterozygous mice resulted in reporter gene expression in the cortical region, hippocampal dentate gyrus (DG) and anterior CA2 (aCA2)/CA3, paraventricular thalamus (PVT), central nucleus of amygdala (CeA), dorsal endopiriform nucleus (DEn), and posteromedial cortical amygdaloid area (PMCo), where endogenous OXTR expression has previously been reported. Compared to reporter expression when breeding the constitutive iCre line (Stock No. 037578) to Ai9 mice, where too many cells are labeled with tdTomato to be able to clearly distinguish each neuron, reporter expression is only sparsely induced in this inducible icre line, allowing the identification of cell morphology at single-cell resolution.

Of note, a tdTomato expressing line is also available as (Stock No. 037580).

The donating investigator reports that this strain, founder line 8, does not contain the ampicillin resistance gene (AMP) cassette that was used in the development of the strain.

Development

crRNA/tracrRNA duplex gRNA (incorporating crRNA CCAUCCUCGGCAUGAGCCAUguuuuagagcuaugcuguuuug) was designed to insert a 3xHA-tag, a viral 2A oligopeptide (T2A; mediates ribosomal skipping), and an icre/ERT2 sequence (a codon improved cre fused to mutated human estrogen receptor ligand binding domain) immediately before the stop codon of the oxytocin receptor (Oxtr) gene on chromosome 6. The crRNA/tracrRNA, targeting vector, and the Cas9 nuclease protein were introduced into the pronuclei of fertilized eggs from hybrid B6C3F1 mice. Targeted embryos were transferred to pseudopregnant females. Correctly targeted pups were identified by PCR and Sanger sequencing and confirmed the absence of random insertions. Resulting Oxtr^{3xHA-iCreERT2} mice were further backcrossed to C57BL/6J mice (Stock No. 000664) for one generation by the donating laboratory. The donating investigator reports that this strain, specifically founder line 8, does not contain the ampicillin resistance gene (AMP) cassette that was used in the development of the strain. Cryopreserved sperm was sent to The Jackson Laboratory. Upon arrival, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes to establish our live colony.

Allele

Symbol: Oxtr^{em4(icre/ERT2)Yinn}
Name: endonuclease-mediated mutation 4, Yukiko U Inoue
MGI accession ID: MGI:7282236
Generation method: Endonuclease-mediated
Attributes: Recombinase-expressing; Inducible; Epitope tag
Synonyms: Oxtr^{3xHA-iCreERT2}; Oxtr^{em2(icre/ERT2)Inot}; Oxtr-T2A-iCre-ERT2
Marker symbol: Oxtr
Marker name: oxytocin receptor
Marker synonyms: OTR; OT-R; OTR1
Chromosome: 6
Strain of origin: (C57BL/6 x C3H)F1
Molecular note: Using CRISPR/cas9 technology, three copies of HA-tag and a T2A-iCre-ERT2 expression cassette were inserted into the Oxtr gene just upstream of the stop codon.