STOCK Agrp^tm1(cre)Lowl/J

Stock number: 012899
Product name: Agrp-Ires-cre
Research areas: Neurobiology Research

Description

AgRP-IRES-Cre knock-in mice have IRES-Cre inserted downstream of the stop codon of the agouti related neuropeptide locus. Cre recombinase expression is observed in agouti-related protein secreting neurons. These mice may be useful for studying the physiological functions of GABA neurotransmission in agouti-related protein neurons in the hypothalamus.

In 2021, sequencing analysis performed by The Jackson Laboratory revealed that the gene targeting used to create the Agrp-Ires-Cre allele also resulted in a duplication of chromosome 8 genomic sequence at least 53 kb in size (including 3 loci) that inserted into intron 1 of the nearby Ripor1 locus. See Detailed Description section for more information.

Detailed description

Mice homozygous for the AgRP-IRES-Cre knock-in allele are viable and fertile, with IRES-Cre inserted downstream of the Agrp stop codon. As such, cre expression is directed by the endogenous promoter/enhancer regions. Specifically, Cre recombinase expression is observed in agouti-related protein (AgRP) secreting neurons. When bred with mice containing a loxP-flanked sequence of interest, Cre-mediated recombination will result in deletion of the floxed sequence(s) in the offspring. These mice may be useful for studying the physiological functions of GABA neurotransmission in ArGP neurons in the hypothalamus.

When AgRP-IRES-Cre mice are bred to have Cre-inducible channelrhodopsin/EYFP expression (e.g., Ai32 [Stock Nos. 012569/024109]), the resulting double mutant animals allow manipulation of AgRP-expressing neuron dynamics (sensory and post-ingestive signals) in response to caloric food and/or experimental compound consumption [Su, Alhadeff and Betley 2017 Cell Rep. 21:2724].

In 2021, sequencing analysis performed by The Jackson Laboratory revealed that the gene targeting used to create the Agrp-Ires-Cre allele also resulted in a duplication of chromosome 8 genomic sequence at least 53 kb in size that inserted into intron 1 of the nearby Ripor1 (Fam65a) locus. The duplicated sequence includes the entire Hsd11b2 locus, the entire Atp6v0d1 locus and at least 11.7 kb of the Agrp locus - likely resulting in overexpression of those genes. While the insertion appears to be in the ~9 kb intron upstream of the translation start site-containing exon 2, the effect on Ripor1 expression (if any) was not specifically analyzed during our investigation.

Development

The AgRP-IRES-Cre knock-in allele was created to have an IRES-Cre inserted after the stop codon of the agouti related neuropeptide locus (Agrp) on chromosome 8. The specific details are below.

The targeting vector contained an internal ribosome entry site (IRES) fused to a Cre recombinase gene, followed by a frt-flanked kanamycin/neomycin (kan/neo) resistance cassette. This was targeted downstream of the Agrp stop codon (originally identified as being in exon 3, but more recent exon numbering refers to this same exon as exon 5). The construct was electroporated into W4-derived 129S6/SvEvTac embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts and resulting chimeric males were bred to C57BL/6 x 129 or FVB females. Offspring were bred with Flp-expressing mice (129S4/SvJaeSor-Gt(ROSA)26Sor^tm1(FLP1)Dym/J [Stock No. 003946]) for germline deletion of the kan/neo cassette. Progeny were crossed to remove the Flp-expressing allele. Mice from the resulting AgRP-IRES-Cre colony were sent to The Jackson Laboratory. Upon arrival, mice were bred to C57BL/6J inbred mice (Stock No. 000664) for at least one generation to establish our live colony.

In 2021, sequencing analysis performed by The Jackson Laboratory revealed that the gene targeting used to create the Agrp-Ires-Cre allele also resulted in a duplication of chromosome 8 genomic sequence at least 53 kb in size that inserted into intron 1 of the nearby Ripor1 (Fam65a) locus. The duplicated sequence includes the entire Hsd11b2 locus, the entire Atp6v0d1 locus and at least 11.7 kb of the Agrp locus - likely resulting in overexpression of those genes. While the insertion appears to be in the ~9 kb intron upstream of the translation start site-containing exon 2, the effect on Ripor1 expression (if any) was not specifically analyzed during our investigation.

Allele

Symbol: Agrp^tm1(cre)Lowl
Name: targeted mutation 1, Bradford B Lowell
MGI accession ID: MGI:3815085
Generation method: Targeted
Attributes: Recombinase-expressing
Marker symbol: Agrp
Marker name: agouti related neuropeptide
Chromosome: 8
Strain of origin: 129S6/SvEvTac
Expressed genes: cre,cre recombinase,bacteriophage P1
Site of expression: agouti-related protein (AgRP) neurons of the brain
Molecular note: An IRES-cre-frt-kan/neo-frt cassette was inserted in frame in exon 5 after the stop codon via homologous recombination. The kan/neo cassette was subsequently removed via in vivo Flp mediated recombination. Crosses to reporter mice detected Cre activity in AGRP+ neurons. The mutation resulted in a duplication of chromosome 8 genomic sequence at least 53 kb in size that inserted into intron 1 of the nearby Ripor1 (Fam65a) locus. The duplicated sequence includes the entire Hsd11b2 locus, the entire Atp6v0d1 locus and at least 11.7 kb of the Agrp locus. The insertion appears to be in the ~9 kb intron upstream of the translation start site-containing exon 2,however, the effect on Ripor1 expression was not specifically analyzed.