B6(Cg)-Calb2^{tm2.1(cre/ERT2)Zjh}/J

Stock number: 013730
Research areas: Neurobiology Research, Research Tools

Description

The Calb2-CreERT2 (CR-CreER or CR-CreERT2) knock-in allele was designed to both abolish Calb2 (calbindin 2; also called calretinin or CR) gene function and direct CreER^T2 fusion protein expression to calretinin positive interneurons in the brain by the endogenous Calb2 promoter/enhancer elements. Cre-ERT2 fusion gene activity is inducible by tamoxifen administration.

Detailed description

The Calb2-CreERT2 (CR-CreER or CR-CreERT2) knock-in allele was designed to both abolish Calb2 (calbindin 2; also called calretinin or CR) gene function and expresses CreER^T2 fusion protein from the Calb2 promoter/enhancer elements. Cre-ERT2 fusion gene activity is inducible; observed following tamoxifen administration. As such, when CR-CreERT2 mice are bred with mice containing loxP-flanked sequences, tamoxifen-inducible Cre-mediated recombination will result in deletion of the floxed sequences in the CR-expressing cells of the offspring.

Heterozygous mice are viable, fertile, normal in size and do not display any gross physical or behavioral abnormalities. The donating investigator reports that the phenotype of CR-CreER homozygous mice has not been assessed. However, CR-CreER homozygotes may be expected to exhibit the same phenotype as mice homozygous for other null mutations of this gene on a similar genetic background (impaired motor coordination, abnormal nervous system electrophysiology, and impaired long-term potentiation induction in dentate gyrus). Calretinin mRNA or protein expression from the CR-CreER mutant allele was not determined. The donating investigator also reports tamoxifen-inducible Cre recombinase activity recapitulates the endogenous calretinin expression pattern, but with moderate efficiency of induction. In olfactory bulb, the induction efficiency is higher. Tamoxifen-inducible Cre recombinase activity is not observed prior to tamoxifen treatment. Following tamoxifen administration, Cre recombinase activity is observed in calretinin positive interneurons in the brain. The donating investigators did not examine cre expression in tissues other than brain.

For characterization information, see images at the Allen Institute for Brain Science website (Calb2-CreERT2 images).

The Cre-ERT2 fusion protein consists of Cre recombinase fused to a triple mutant form of the human estrogen receptor which does not bind its natural ligand (17β-estradiol) at physiological concentrations but will bind the synthetic estrogen receptor ligands 4-hydroxytamoxifen (OHT or tamoxifen) and, with lesser sensitivity, ICI 182780. Restricted to the cytoplasm, Cre-ERT2 can only gain access to the nuclear compartment after exposure to tamoxifen. To counteract the mixed estrogen agonist effects of tamoxifen injections, which can result in late fetal abortions in pregnant mice, progesterone may be coadministered.

Development

A targeting vector was designed to insert a CreER^T2 fusion gene (Cre-ER^T2; Cre recombinase fused to a G400V/M543A/L544A triple mutation of the human estrogen receptor ligand binding domain), an SV40 polyA signal, and an frt-flanked neo cassette into the initiation codon of the Calb2 (calbindin 2; also called calretinin or CR) locus. This construct was electroporated into C57BL/6-derived Bruce4 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts and chimeric mice were bred with white C57BL/6 mice (harboring the Tyr^c-2J mutation) to originate the colony. Mutant mice were bred with Actin-FLPe mice (on a C57BL/6 congenic background (N10); see Stock No. 005703) to remove the neo selection cassette. These Calb2-CreERT2 (CR-CreER or CR-CreERT2) mice were subsequently bred to white C57BL/6 mice for a few additional generations (and the FLPe transgene was removed) prior to sending to The Jackson Laboratory Repository. Upon arrival, mice were bred with C57BL/6J inbred mice (Stock No. 000664) for at least one generation to establish the Calb2-CreERT2 colony. These Calb2-CreERT2 mice may still be segregating at the Tyr locus.

Allele

Symbol: Calb2^{tm2.1(cre/ERT2)Zjh}
Name: targeted mutation 2.1, Z Josh Huang
MGI accession ID: MGI:4880758
Generation method: Targeted
Attributes: Recombinase-expressing; Inducible
Marker symbol: Calb2
Marker name: calbindin 2
Chromosome: 8
Strain of origin: C57BL/6
Expressed genes: cre/ERT2,Cre recombinase and estrogen receptor 1 (human) fusion gene,
Site of expression: Following tamoxifen administration, Cre recombinase activity is observed in calretinin positive interneurons in the brain.
Molecular note: A targeting vector was designed to insert a CreERT2 fusion gene, an SV40 polyA signal, and anFRT-flanked neo cassette into the initiation codon of the Calb2 (calbindin 2; also called calretinin or CR) locus. This construct was electroporated into C57BL/6-derived embryonic stem (ES) cells. Mutant mice were bred with Actin-FLPe mice to remove the neo cassette,