B6;129S-Cartpt^{tm1.1(cre)Hze}/J

Stock number: 028533
Product name: Cart-IRES2-Cre-D
Research areas: Cancer Research, Neurobiology Research, Research Tools

Description

Cart-IRES2-Cre-D knockin mice have Cre recombinase expression directed to CART prepropeptide-expressing cells, without disrupting endogenous Cartpt expression. These mice may be used to generate conditional mutations for studying gain-or-loss of function and/or fate mapping in subgroups of brain tissues (including cortex, cerebral nuclei, hypothalamus and midbrain) in regards to feeding, appetite and stress response, as well as gene regulation by cocaine and other drugs.

Detailed description

The Cartpt gene encodes CART prepropeptide isoforms that are processed into multiple biologically active peptides. Expression of this gene is regulated by cocaine and other drugs, and is associated with feeding/appetite and stress response.
The Cart-IRES2-Cre-D knockin mutation has an IRES2 sequence and a Cre recombinase gene inserted downstream of the translational STOP codon of the Cartpt gene. As such, Cart-IRES2-Cre-D mice have both endogenous gene and Cre recombinase expression directed to CART prepropeptide-expressing cells by the endogenous promoter/enhancer elements of the Cartpt locus. When Cart-IRES2-Cre-D mice are bred with mice containing loxP-flanked sequences, Cre-mediated recombination will result in deletion of the floxed sequences in Cartpt-expressing cells in the offspring.

The donating investigator reports Cre recombinase expression and activity largely recapitulates the endogenous Cartpt gene's unique expression pattern in areas such as cortex, cerebral nuclei, hypothalamus and midbrain.

The donating investigator did not examine cre expression in tissues other than brain.

Heterozygous mice are viable and fertile with no reported gross physical or behavioral abnormalities. To date (December 2015), it has not been attempted to make this strain homozygous.

Development

These Cart-IRES2-Cre-D mice (also called Cart-IRES2-Cre-Δ or Cart-IRES2-Cre-Δneo) have an IRES2 sequence and a Cre recombinase gene inserted immediately downstream of the translational STOP codon of the CART prepropeptide gene (Cartpt) on chromosome 13. The specific details are below.

The targeting vector contained, from 5' to 3', a partial Cartpt sequence spanning exon 1 through intron 2, an frt3 site within Cartpt intron 2, a partial Cartpt exon 3 sequence up to and including the endogenous stop codon, an internal ribosome entry site 2 (IRES2) sequence (allows translation initiation in the middle of an mRNA sequence), a Cre recombinase gene, a bovine growth hormone polyA sequence, an AttB site, a PGK promoter-Neomycin resistance gene-PGK polyA cassette, an frt5 site, an mRNA splice acceptor sequence, the 3' portion of the hygromycin gene (Hygro2) with SV40 late polyA signal, and an AttP site.

This construct was electroporated into (129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts. Chimeric mice were bred to PhiC31-expressing mice (C57BL/6J congenic background; see Stock No. 007743) to remove the AttB/AttP-flanked sequences (PGK-Neo-polyA::frt5::RNA splice acceptor::3'hygro-polyA) and replace it with the recombined AttB/AttP site (AttL).

The resulting Cart-IRES2-Cre-D mice were bred with C57BL/6J wildtype mice for several generations (and the PhiC31 transgene was removed). In 2016, black male mice from generation N4 were sent to The Jackson Laboratory Repository. Upon arrival, males are used to cryopreserve sperm. To establish the living Cartpt-IRES2-Cre-D mouse colony, an aliquot of the frozen sperm is used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).

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

Allele

Symbol: Cartpt^{tm1.1(cre)Hze}
Name: targeted mutation 1.1, Hongkui Zeng
MGI accession ID: MGI:5704439
Generation method: Targeted
Attributes: Recombinase-expressing
Marker symbol: Cartpt
Marker name: CART prepropeptide
Chromosome: 13
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: cre,cre recombinase,bacteriophage P1
Site of expression: Cre recombinase expression recapitulates the endogenous Cartpt gene's expression in the cortex, cerebral nuclei, hypothalamus and midbrain.
Molecular note: The targeting vector contains, from 5' to 3', a partial Cartpt sequence spanning exon 1 through intron 2, an FRT3 site within Cartpt intron 2, a partial Cartpt exon 3 sequence up to and including the endogenous stop codon, an internal ribosome entry site 2 (IRES2) sequence (allows translation initiation in the middle of an mRNA sequence), a Cre recombinase gene, a bovine growth hormone polyA sequence, an AttB site, a PGK promoter-Neomycin resistance gene-PGK polyA cassette, an FRT5 site, an mRNA splice acceptor sequence, the 3' portion of the hygromycin gene (Hygro2) with SV40 late polyA signal, and an AttP site. PhiC31-mediated recombination removed the AttB/AttP-flanked sequence (PGK-Neo-polyA::frt5::RNA splice acceptor::3'hygro-polyA) and replaced it with the recombined AttB/AttP site (AttL).