B6.Cg-Nxph4^{tm1.1(cre/ERT2)Hze}/J

Stock number: 022861
Product name: Nxph4-2A-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 neurexophilin 4 locus. When induced, cre activity is observed in cortical layer 6b neurons and other scattered small cells [possibly glia] throughout the cortex.

Detailed description

Mice heterozygous for the Nxph4-2A-CreERT2-D targeted mutation are viable and fertile. The Nxph4-2A-CreERT2-D targeted mutation has a viral 2A oligopeptide (T2A) that mediates ribosomal skipping and a CreER^T2 fusion gene (CreERT2 fusion protein) inserted downstream of the neurexophilin 4 translational STOP codon.

As such, Nxph4-2A-CreERT2-D mice have both endogenous gene and CreERT2 fusion protein expression directed to Nxph4-expressing cells by the endogenous promoter/enhancer elements of the neurexophilin 4 locus. CreERT2 fusion gene activity is inducible; observed following tamoxifen administration. When Nxph4-2A-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 Nxph4-expressing cells of the double mutant offspring.

Specifically, the donating investigator reports that following tamoxifen induction, Nxph4-2A-CreERT2-D directs reporter gene expression to cortical layer 6b neurons and in other scattered small cells (possibly glia) throughout the cortex, in a pattern that partially recapitulates the endogenous Nxph4 expression.

The donating investigator reports no CreERT2 activity (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 (Nxph4-2A-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 Nxph4-2A-CreERT2-D targeted mutation (also called Nxph4-2A-CreERT2-Δ or Nxph4-2A-CreERT2-Δneo) has a viral 2A oligopeptide (T2A) that mediates ribosomal skipping and a CreER^T2 fusion gene inserted immediately downstream of the neurexophilin 4 translational STOP codon. The specific details are below.

The targeting vector contained, from 5' to 3', a partial Nxph4 intron sequence containing an frt3 site, a partial Nxph4 exon 2 sequence up to (but not including) the endogenous stop codon, a T2A sequence that is in-frame with the Nxph4 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 Nxph4 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, and an AttP site.

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 Nxph4-2A-CreERT2-D mice were bred with C57BL/6J wildtype mice for several generations (and the PhiC31 transgene was removed) prior to sending generation N5F1 males to The Jackson Laboratory Repository in 2013. Upon arrival, males were used to cryopreserve sperm. To establish the living Nxph4-2A-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: Nxph4^{tm1.1(cre/ERT2)Hze}
Name: targeted mutation 1.1, Hongkui Zeng
MGI accession ID: MGI:5501233
Generation method: Targeted
Attributes: Recombinase-expressing; Inducible
Marker symbol: Nxph4
Marker name: neurexophilin 4
Chromosome: 10
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: Following tamoxifen induction, reporter gene expression is found in cortical layer 6b neurons and in other scattered small cells (possibly glia) throughout the cortex.
Molecular note: A targeting vector contained a partial Nxph4 intron sequence containing an frt3 site, a partial Nxph4 exon 2 sequence up to (but not including) the endogenous stop codon, a self-cleavingT2A 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, and an AttP site. 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).