B6;129S-Calb1^{tm2.1(cre)Hze}/J

Stock number: 028532
Product name: Calb1-IRES2-Cre-D
Research areas: Cancer Research, Neurobiology Research, Research Tools

Description

Calb1-IRES2-Cre-D knockin mice are designed to have Cre recombinase expression directed to calbindin 1-expressing cells in the hippocampus, striatum, thalamus, midbrain and cerebellum, without disrupting endogenous Calb1 expression. These mice may be used to generate conditional mutations for studying gain-or-loss of function and/or fate mapping related to this calcium-binding protein.

Detailed description

The Calb1-IRES2-Cre-D knockin mutation has an IRES2 sequence and a Cre recombinase gene inserted downstream of the translational STOP codon of the calbindin 1 gene (Calb1). As such, Calb1-IRES2-Cre-D mice are designed to have both endogenous gene and Cre recombinase expression directed to calbindin 1-expressing cells by the endogenous promoter/enhancer elements of the Calb1 locus. When Calb1-IRES2-Cre-D mice are bred with mice containing lox-flanked sequences, Cre-mediated recombination will result in deletion of the floxed sequences in Calb1-expressing cells in the offspring. The donating investigator reports Cre recombinase expression and activity largely recapitulates the endogenous Calb1 gene's unique expression pattern in many parts of the brain, such as cortex, hippocampus, striatum, thalamus, midbrain and cerebellum. The donating investigator did not examine cre expression in tissues other than brain. Heterozygous and homozygous mice are viable and fertile with no reported gross physical or behavioral abnormalities.

Development

These Calb1-IRES2-Cre-D mice (also called Calb1-IRES2-Cre-Δ or Calb1-IRES2-Cre-Δneo/hygro) have an IRES2 sequence and a Cre recombinase gene inserted immediately downstream of the translational STOP codon of the calbindin 1 gene (Calb1) on chromosome 4. The specific details are below.

The targeting vector contained, from 5' to 3', a partial Calb1 sequence spanning intron 6 through intron 10, an frt3 site within Calb1 intron 10, a partial Calb1 exon 11 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 Calb1-IRES2-Cre-D mice were bred with C57BL/6J wildtype mice for several generations (and the PhiC31 transgene was removed). In 2016, male mice from generation N5 with black coat color were sent to The Jackson Laboratory Repository. Upon arrival, males are used to cryopreserve sperm. To establish the living Calb1-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).

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 rederived living colony at The Jackson Laboratory Repository. Two of the 43 markers, on Chromosomes 11 and 15 were segregating, suggesting an incomplete backcross.

Allele

Symbol: Calb1^{tm2.1(cre)Hze}
Name: targeted mutation 2.1, Hongkui Zeng
MGI accession ID: MGI:5792816
Generation method: Targeted
Attributes: Recombinase-expressing
Marker symbol: Calb1
Marker name: calbindin 1
Chromosome: 4
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: cre,cre recombinase,bacteriophage P1
Site of expression: Cre recombinase expression and activity largely recapitulates the endogenous Calb1 gene's unique expression pattern in many parts of the brain, such as cortex, hippocampus, striatum, thalamus, midbrain and cerebellum.
Molecular note: The targeting vector contains, from 5' to 3', an FRT3 site within Calb1 intron 10, a partial Calb1 exon 11 sequence up to and including the endogenous stop codon,an IRES2 sequence, Cre recombinase, a polyA sequence, an AttB site, a PGK-Neomycin 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). Mice have both endogenous gene and Cre recombinase expression directed to calbindin 1-expressing cells by the endogenous promoter/enhancer elements of the Calb1 locus.