While heterozygous mice are viable and fertile with no reported abnormalities, homozygous mice die shortly after birth. The Six2GCE "knock-in" allele both abolishes Six2 gene function and expresses an eGFPCreERT2 fusion protein (EGFP and creERT2 fusion protein) from the Six2 promoter/enhancer elements. While EGFP immunofluorescence is observed in nephron progenitor population cap mesenchyme from the onset of metanephric kidney development, Cre-ERT2 fusion gene activity is inducible; observed in the same cells only following tamoxifen administration. As such, when Six2GCE mice are bred with mice containing loxP-flanked sequence, tamoxifen-inducible, Cre-mediated recombination will result in deletion of the floxed sequences in the Six2-expressing cells of the offspring.
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.
