B6.Cg-Penk^{tm1.1(cre/ERT2)Hze}/J

Stock number: 022862
Product name: Penk-2A-CreERT2-D
Research areas: Cancer Research, Neurobiology Research, Research Tools

Description

These knockin mice express a tamoxifen-inducible cre recombinase from the endogenous promoter/enhancer elements of the preproenkephalin locus. When induced, cre activity is observed in the striatum, olfactory tubercle, and sparse cells in the dentate gyrus and cortex.

Detailed description

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 CreER^T2 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.

Development

The Penk-2A-CreERT2-D targeted mutation (also called Penk-2A-CreERT2-Δ or Penk-2A-CreERT2-Δneo) has a CreER^T2 fusion gene inserted downstream of the preproenkephalin translational STOP codon, with both genes separated by an in-frame F2A peptide cleavage signal (to promote expression of both individual genes). The specific details are below.

The targeting vector contained, from 5' to 3', a partial Penk intron 1 sequence containing an frt3 site, a partial Penk exon 2 sequence up to (but not including) the endogenous stop codon, a foot-and-mouth disease virus self-cleaving 2A peptide sequence (FMDV 2A or F2A; to promote individual expression the genes before and after it) that is in-frame with the Penk coding sequence, a CreER^T2 fusion gene (Cre-ER^T2; Cre recombinase fused to a G400V/M543A/L544A triple mutation of the human estrogen receptor ligand binding domain) that is in-frame with the Penk coding sequence, a bovine growth hormone polyA signal, an AttB site, a PGK-Neo-polyA cassette, an frt5 site, an mRNA splice acceptor, the 3' portion of the hygromycin gene with SV40 polyA signal, an AttP site, and genomic sequence from the Penk locus starting 509 bp downstream of the endogenous stop codon (to create a 511 bp deletion downstream of, and including, the stop codon).

This targeting vector was electroporated into (129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts. The resulting chimeric animals were bred to PhiC31-expressing mice (C57BL/6J congenic background; see Stock No. 007743) to delete the AttB/AttP-flanked sequences (PGK-neo-polyA::frt5::mRNA splice acceptor::3'hygro::SV40 polyA) and replace it with the recombined AttB/AttP site (AttL). The resulting Penk-2A-CreERT2-D mice were bred with C57BL/6J wildtype mice for several generations (and the PhiC31 transgene was removed) prior to sending generation N7F1 males to The Jackson Laboratory Repository in 2013. During backcrossing, the Y chromosome may not have been fixed to the C57BL/6J genetic background. Upon arrival, males were used to cryopreserve sperm. To establish the living Penk-2A-CreERT2-D mouse colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).

Allele

Symbol: Penk^{tm1.1(cre/ERT2)Hze}
Name: targeted mutation 1.1, Hongkui Zeng
MGI accession ID: MGI:5501235
Generation method: Targeted
Attributes: Recombinase-expressing; Inducible
Marker symbol: Penk
Marker name: preproenkephalin
Chromosome: 4
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: cre/ERT2,Cre recombinase and estrogen receptor 1 (human) fusion gene,
Site of expression: Cre recombinase expression (in situ hybridization) is strong in striatum, in olfactory tubercle, and in sparse dentate gyrus and cortex, and is observed weakly throughout the brain.
Molecular note: A targeting vector contained a partial Penk intron 1 sequence containing an frt3 site, a partial Penk exon 2 sequence up to (but not including) the endogenous stop codon, a self-cleaving T2A sequence in-frame with the Nxph4 coding sequence, a cre/ERT2 fusion gene, a bovine growth hormone polyA signal, an AttB site, a PGK-Neo-polyA cassette, an frt5 site, an mRNA splice acceptor, the 3' portion of the hygromycin gene with SV40 polyA signal, an AttP site and genomic sequence from the Penk locus starting 509 bp downstream of the endogenous stop codon (to create a 511 bp deletion including, the stop codon). This targeting vector was electroporated into G4 ES cells. Correctly targeted ES cells were injected into recipient blastocysts. The resulting chimeric animals were bred to PhiC31-expressing mice to delete the AttB/AttP-flanked sequences and replace it with the recombined AttB/AttP site (AttL).