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-ERT2 [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.
