B6;129-Cckbr^{tm1.1(cre)Mgmj}/J

Stock number: 036749
Product name: Cckbr-cre
Research areas: Cancer Research, Diabetes and Obesity Research, Metabolism Research, Neurobiology Research, Research Tools

Description

Cckbr^cre knock-in mice have the endogenous cholecystokinin B receptor promoter/enhancer sequences directing expression of Cre recombinase. These mice are a Cre-lox tool allowing Cre recombination in Cckbr-expressing cells/tissues (including neurons of the ventromedial nucleus of the hypothalamus), and may be useful in studying feeding behavior, obesity and diabetes.

Detailed description

The Cckbr gene encodes the cholecystokinin B receptor, which is associated with the insulin-independent homeostatic control of blood glucose, as well as susceptibility to endotoxin shock, and pain and and touch sensitivity.

The Cckbr^cre knock-in allele has a T2A sequence and a Cre recombinase gene inserted downstream of the Cckbr translational STOP codon. This is designed to have the endogenous Cckbr promoter/enhancer elements directing cre expression primarily in neurons of the ventromedial nucleus of the hypothalamus (VMN; see below), as well as to retain endogenous Cckbr expression.

The donating investigator reports that when crossed with GFP-expressing Cre-reporter mice (such as Stock No. 024750), GFP expression can be observed in Cckbr-expressing neurons of the VMN, with some sparse labeling in the dorsomedial hypothalamus, recapitulating the pattern of endogenous Cckbr expression.
Moreover, following intra-VMN administration of AAV-DIO-ChR2-eYFP, optogenetic activation of Cckbr^cre-expressing neurons by 473nm laser stimulation (following a period of 4-5 hours food deprivation) results in increased blood glucose levels, as well as increased plasma levels of epinephrine, norepinephrine, and corticosterone (Flak et al., 2020).

Development

The Cckbr-Cre knock-in allele (also called Cckbr^cre) was created in the laboratory of Dr. Martin Myers (University of Michigan) to have a T2A sequence and a Cre recombinase gene inserted immediately downstream of the cholecystokinin B receptor locus (Cckbr) on chromosome 7. The specific details are below.

The targeting vector contained, from 5' to 3', a viral 2A oligopeptide sequence (T2A; mediates ribosomal skipping), a Cre recombinase gene, a bovine growth hormone polyA sequence, an FRT site, a PGK-neo-polyA cassette and an FRT site. The construct was electroporated into (129X1/SvJ x 129S1/Sv)F1-Kitl⁺-derived R1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts, and chimeric mice were bred with C57BL/6J for germline transmission. Offspring were bred with Flpo-expressing mice (ROSA26-FLPo on a C57BL/6J background; Stock No. 012930) for germline deletion of the PGK-neo-polyA cassette. The resulting Cckbr^cre colony was then backcrossed to C57BL/6J mice for at least 3 generations (and the Flpo-expressing allele was removed) prior to sending males to The Jackson Laboratory Repository in 2022 (see SNP note below). Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

Of note, the donating investigator reports that, at least once during backcrossing, a Cckbr^cre female was bred to a C57BL/6J inbred male (thus the Y chromosome of the congenic strain is of C57BL/6J origin).

In 2022, 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. Six of the 43 markers throughout the genome were segregating with 129, suggest an incomplete backcross.

Allele

Symbol: Cckbr^{tm1.1(cre)Mgmj}
Name: targeted mutation 1.1, Martin G Myers, Jr
MGI accession ID: MGI:6834787
Generation method: Targeted
Attributes: Recombinase-expressing
Synonyms: Cckbr^Cre
Marker symbol: Cckbr
Marker name: cholecystokinin B receptor
Marker synonyms: CCK2/gastrin; Cck2r; CCK-2R; CCK-B; CCK-B/gastrin receptor; CCKR-2; Cholrec; GASR
Chromosome: 7
Strain of origin: (129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Molecular note: Using homologous recombination in ES cells, a T2A sequence and a CreER(T2) fusion gene were inserted immediately downstream of the cholecystokinin B receptor locus (Cckbr) on chromosome 7. The targeting vector contained, from 5' to 3', a viral 2A oligopeptide sequence (T2A; mediates ribosomal skipping), a Cre recombinase gene, a bovine growth hormone polyA sequence, an frt site, a PGK-neo-polyA cassette, and an frt site. Founder mice were bred with Flpo-expressing mice to delete the PGK-neo-polyA cassette.