B6(129S4)-Et(icre/ERT2)14208Rdav/J

Stock number: 012690
Research areas: Neurobiology Research, Research Tools

Description

These mice express the tamoxifen-inducible iCre-ERT2 fusion protein (iCre-ER^T2) under the control of the promoter/enhancer regions surrounding the site where the enhancer trap transgene randomly inserted. These mice may be useful for generating conditional mutations for studying gain or loss of function and/or fate mapping in brain tissues.

Detailed description

Mice hemizygous for this enhancer trap lentiviral transgene are viable and fertile, with expression of an iCreERT2 fusion gene (an optimized variant of Cre recombinase [iCre; improved with mammalian codon usage, removed putative cryptic splice sites, altered stop codon, and reduced CpG content to limit the chances of epigenetic silencing in mammals] fused to Cre-ER^T2 [a G400V/M543A/L544A triple mutation of the human estrogen receptor ligand binding domain]) under control of the promoter/enhancer regions surrounding the site where the enhancer trap lentiviral transgene randomly inserted. Cre-ERT2 fusion gene activity is designed to be inducible (observed following tamoxifen administration).

Specifically, the donating investigator reports Cre recombinase activity in brain tissues as: scattered cells in the neocortex, hippocampus, amygdala, striatum, thalamus, hypothalamus, midbrain, pons, medulla, and many cerebellar granule cells. No Cre recombinase activity is observed prior to tamoxifen exposure, and no Cre recombinase activity is observed in other tested tissues.

When these enhancer trap lentiviral transgenic mice are bred with mice containing loxP-flanked sequences, tamoxifen-inducible Cre-mediated recombination will result in deletion of the floxed sequences in the cre-expressing cells of the double mutant offspring.

For characterization information, see images at the Allen Institute for Brain Science website (Et(icre/ERT2)14208Rdav 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

An enhancer trap lentiviral transgenic approach was used to generate these mice. The FUGW-hsyn-iCreERT2-WPRE transgene was designed with a self-inactivating lentiviral vector backbone (FUGW), a human synapsin minimal promoter (-244 to +47), an iCreERT2 fusion gene (an optimized variant of Cre recombinase [iCre; improved with mammalian codon usage, removed putative cryptic splice sites, altered stop codon, and reduced CpG content to limit the chances of epigenetic silencing in mammals] fused to Cre-ER^T2 [a G400V/M543A/L544A triple mutation of the human estrogen receptor ligand binding domain]), and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability).

This packaged lentivirus was injected subzonally (under the zona pellucida) into 1-2 cell stage C57BL/6J mouse embryos.

Founder mice from line 14208 were bred to B6.R26-lacZ mutant mice (Gt(ROSA)26Sor^tm1Sor mutant mice backcrossed to C57BL/6 for at least 10 generations; similar to Stock No. 003474).

The donating investigator stated that the colony was then maintained by breeding transgenic mice with C57BL/6 or B6.R26-lacZ mice each generation (see SNP note below). At some time after this, transgenic males were bred with C57BL/6 females to cryopreserve embryos, and these embryos were sent to The Jackson Laboratory Repository. Upon arrival, embryos were used to generate living mice. To maintain the live colony (and remove the R26-lacZ mutation), transgenic mice were bred together, to wildtype (noncarrier) mice from the colony, or to C57BL/6J inbred mice (Stock No. 000664).

The donating investigator reports that the transgene insertion site for line 14208 is located in an intron of the apolipoprotein M locus (Apom) on chromosome 17 (35.27 Mbp). No information was provided on whether or not the transgene insertion site disables the Apom 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, at least 1 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: Et(icre/ERT2)14208Rdav
Name: enhancer trap 14208, Ron Davis
MGI accession ID: MGI:4947569
Generation method: Transgenic
Attributes: Recombinase-expressing; Inducible
Marker symbol: Et(icre/ERT2)14208Rdav
Marker name: enhancer trap 14208, Ron Davis
Chromosome: UN
Strain of origin: C57BL/6J
Expressed genes: cre/ERT2,Cre recombinase and estrogen receptor 1 (human) fusion gene,
Site of expression: Cre recombinase activity is observed in the following brain tissues: scattered cells in the neocortex, hippocampus, amygdala, striatum, thalamus, hypothalamus, midbrain, pons, medulla, and many cerebellar granule cells.
Molecular note: An enhancer trap lentiviral transgenic approach was used to generate these mice. The transgene was designed with a self-inactivating lentiviral vector backbone (FUGW), a human synapsin minimal promoter (-244 to +47), a beta-globin intron, an icre /ERT2 fusion (icre; improved with mammalian codon usage, no putative cryptic splice sites, altered stop codon, and reduced CpG content to limit the chances of epigenetic silencing in mammals), fused to a mutated human estrogen receptorligand binding domain (ERT2), and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability. This packaged lentivirus was injected subzonally (under the zona pellucida) into 1-2 cell stage C57BL/6J mouse embryos. Founders were crossed to C57BL/6 animals. Animals were crossed to B6-Gt(ROSA)26Sor^tm1Sor mice to assess cre activity/transmission. Cre positive, lacZ negative mice were subsequently bred to C57BL/6 mice to create the 14208 line.