STOCK Et(icre/ERT2)14374Rdav/J

Stock number: 012691
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. While Cre-ERT2 fusion gene activity is designed to be inducible (observed following tamoxifen administration), some minor Cre recombinase activity is observed without tamoxifen in these mice.

Specifically, the donating investigator reports Cre recombinase activity in brain tissues as: many cells in hippocampus (including dentate gyrus projections), with very sparse coverage in all other areas (olfactory bulb, cortex, striatum, thalamus, midbrain, pons, medulla, and hypothalamus). 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)14374Rdav 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 FVB/N mouse embryos.

Founder mice from line 14374 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 colony was then maintained by breeding transgenic mice with C57BL/6 or B6.R26-lacZ mice each generation. 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 14374 is located at 55.69 Mbp on chromosome 18. Currently, there are no known genes or expressed RNA sequences that have been mapped to this region of the genome. The insertion site is approximately 30 kbp distal to the expressed sequence tag (EST) BU936969. The closest known gene is the predicted gene Gm4221 locus (expressed RNA sequence AK163160), which is approximately 460 kbp proximal to the insertion site.

Allele

Symbol: Et(icre/ERT2)14374Rdav
Name: enhancer trap 14374, Ron Davis
MGI accession ID: MGI:4947570
Generation method: Transgenic
Attributes: Recombinase-expressing; Inducible
Marker symbol: Et(icre/ERT2)14374Rdav
Marker name: enhancer trap 14374, Ron Davis
Chromosome: UN
Strain of origin: FVB/N
Expressed genes: cre/ERT2,Cre recombinase and estrogen receptor 1 (human) fusion gene,
Site of expression: Cre recombinase activity in brain tissues is found in many cells of the hippocampus (including dentate gyrus projections), with very sparse coverage in all other areas (olfactory bulb, cortex, striatum, thalamus, midbrain, pons, medulla, and hypothalamus).
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 14374 line.