C57BL/6J-Crhbp^{em1(cre)Sleep}/J

Stock number: 041667
Product name: Crhbp-Cre
Strain synonyms: Crhbp-2A-Cre-pA
Research areas: Cancer Research, Neurobiology Research, Research Tools

Description

Crhbp-Cre knock-in mice have P2A-Cre inserted downstream of the stop codon of the corticotropin releasing hormone binding protein (Crhbp) gene. Cre recombinase expression is observed in Crhbp expressing neurons. These mice may be useful for studying the physiological functions of neuronal circuits originating in the brainstem, pons, and medulla.

Detailed description

Mice homozygous for the Crhbp-Cre knock-in allele are viable and fertile, with P2A-Cre inserted downstream of the Crhbp stop codon. Cre expression is directed by the endogenous Crhbp promoter/enhancer regions. Specifically, Cre recombinase expression is observed in the corticotropin releasing hormone binding protein (CRHBP) expressing neurons. When coupled with a Cre-inducible marker injected into a specific region of the brain, or crossed to a mouse with a floxed allele, Cre-mediated recombination will result in deletion of the loxP-flanked sequence. These mice may be useful for studying the neuronal circuits originating in the brainstem, pons, and medulla during rapid eye movement (REM) sleep and dysregulation in Parkinson's disease patients.

Development

Using CRISPR/Cas9 technology, P2A-Cre-pA was inserted immediately before the stop codon of Crhbp gene (reference sequence: ENSMUST00000221025.3 Ensembl Canonical Crhbp-202, 2188bp, 322aa). The targeted sequence for CRISPR/Cas9 was 5'-CTGTTTGTCTAGTCTGTGAATGG-3'. A donor plasmid containing P2A-Cre-pA flanked by 5'- and 3'-homology arms (1.4 kbp each) and plasmid pX33054 carrying Cas9 and the gRNA were microinjected into C57BL/6J zygotes. A progeny with an insertion of a single copy of Cre was determined by quantitative PCR. A line exhibiting germline transmission of the transgene, confirmed by natural mating with wildtype C57BL/6J mice, was established. Finally, the expected transgene insertion by PCR and the single-copy insertion of Cre described above were confirmed again in the F1 generation. The resultant mice were backcrossed to C57BL/6J for at least 4 generations. The heterozygotes were then intercrossed to generate homozygous mice and maintained on a C57BL/6J genetic background by the donating laboratory. Upon arrival at The Jackson Laboratory, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

Allele

Symbol: Crhbp^{em1(cre)Sleep}
Name: endonuclease-mediated mutation 1, Yu Hayashi
MGI accession ID: MGI:8249410
Generation method: Endonuclease-mediated
Attributes: Recombinase-expressing
Synonyms: Crhbp-2A-Cre
Marker symbol: Crhbp
Marker name: corticotropin releasing hormone binding protein
Marker synonyms: CRFBP; CRF-BP; CRH-BP
Chromosome: 13
Strain of origin: C57BL/6J
Molecular note: CRISPR/Cas9 genome editing technology was used to insert a P2A-Cre-pA cassette immediately before the stop codon of the gene.