B6(Cg)-Etv1^{tm1.1(cre/ERT2)Zjh}/J

Stock number: 013048
Product name: ER81-CreER
Research areas: Neurobiology Research, Research Tools

Description

The Etv1-CreERT2 knock-in allele was designed to both abolish ets variant gene 1 (Etv1) gene function and direct CreER^T2 fusion protein expression to ER81 positive neurons (including Layer5 pyramidal neurons) by the endogenous Etv1 promoter/enhancer elements. Cre-ERT2 fusion gene activity is inducible by tamoxifen administration.

Detailed description

The ER81-CreER (or ER81-CreERT2, Etv1-CreER, Etv1-CreERT2) knock-in allele was designed to both abolish ets variant gene 1 (Etv1) gene function and expresses CreER^T2 fusion protein from the Etv1 promoter/enhancer elements. Cre-ERT2 fusion gene activity is inducible; observed following tamoxifen administration. As such, when ER81-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 Etv1-expressing cells of the offspring.

Heterozygous mice are viable, fertile, normal in size and do not display any gross physical or behavioral abnormalities. Although mice homozygous for other null mutations of this gene on a similar genetic background exhibit neuromuscular abnormalities, ataxia and premature lethality, the donating investigator reports that ER81-CreER homozygous mice show no gross abnormalities. ER81 mRNA or protein expression from the ER81-CreER mutant allele was not determined. The donating investigator also reports tamoxifen-inducible Cre recombinase activity recapitulates the endogenous Etv1 expression pattern, with moderately efficient inducibility. Tamoxifen-inducible Cre recombinase activity is not observed prior to tamoxifen treatment. Following tamoxifen administration, Cre recombinase activity is observed in ER81 positive cortical neurons (including Layer5 pyramidal neurons). 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 (Etv1-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 ets variant gene 1 locus (Etv1). 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 ER81-CreER (or ER81-CreERT2, Etv1-CreER, Etv1-CreERT2) mice were subsequently bred to C57BL/6 inbred mice for a few additional generations (and the FLPe transgene was removed) prior to sending to The Jackson Laboratory Repository (see SNP note below). Upon arrival, mice were bred with C57BL/6J inbred mice (Stock No. 000664) for at least one generation to establish the ER81-CreERT2 colony. These ER81-CreERT2 mice may still be segregating at the Tyr locus.

A 32 SNP (single nucleotide polymorphism) panel analysis, with 27 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. While the 27 markers throughout the genome suggested a C57BL/6 genetic background, 3 of 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6J ; C57BL/6N genetic background.

Allele

Symbol: Etv1^{tm1.1(cre/ERT2)Zjh}
Name: targeted mutation 1.1, Z Josh Huang
MGI accession ID: MGI:4838417
Generation method: Targeted
Attributes: Recombinase-expressing; Inducible
Marker symbol: Etv1
Marker name: ets variant 1
Chromosome: 12
Strain of origin: C57BL/6
Expressed genes: cre/ERT2,Cre recombinase and estrogen receptor 1 (human) fusion gene,
Site of expression: Cre recombinase activity is observed in ER81 positive cortical neurons (including Layer5 pyramidal neurons).
Molecular note: A targeting vector was designed to insert a Cre/ERT2 fusion gene , an SV40 polyA signal, and an frt-flanked neo cassette into the initiation codon of the ets variant gene 1 locus (Etv1). This construct was electroporated into C57BL/6-derived 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 Tyrc-2J mutation) to originate the colony. Mutant mice were bred with Actin-FLPe mice (on a C57BL/6 congenic background) to remove the neo selection cassette.