Mice heterozygous for the Penk-2A-CreERT2-D targeted mutation (also called Penk-2A-CreERT2-Δ or Penk-2A-CreERT2-Δneo) are viable and fertile. The Penk-2A-CreERT2-D targeted mutation has an in-frame F2A peptide cleavage signal and a CreERT2 fusion gene (CreERT2 fusion protein) inserted downstream of the preproenkephalin translational STOP codon.
As such, Penk-2A-CreERT2-D mice have both endogenous gene and CreERT2 fusion protein expression directed to Penk-expressing cells by the endogenous promoter/enhancer elements of the preproenkephalin locus. CreERT2 fusion gene activity is inducible; observed following tamoxifen administration. When Penk-2A-CreERT2-D mice are bred with mice containing loxP-flanked sequences, tamoxifen-inducible Cre-mediated recombination will result in deletion of floxed sequences in the Penk-expressing cells of the double mutant offspring.
Specifically, the donating investigator reports that Cre recombinase expression (in situ hybridization) is strong in striatum, in olfactory tubercle, and in sparse dentate gyrus and cortex. Furthermore, Cre recombinase expression (in situ hybridization) is observed weakly throughout the brain. Tamoxifen-inducible Cre recombinase activity recapitulates the endogenous Penk expression pattern, but with a relatively low efficiency of induction. The donating investigator reports no Cre recombinase expression (no reporter gene expression) is observed prior to tamoxifen exposure. The donating investigator did not examine CreERT2 activity in tissues other than brain, and did not attempt to generate homozygous mice to date (July 2013).
For characterization information, see images at the Allen Institute for Brain Science website (Penk-2A-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.
