B6.Cg-Fezf1^{tm1.1(cre/folA)Hze}/J

Stock number: 025110
Product name: Fezf1-2A-dCre-D
Research areas: Cancer Research, Neurobiology Research, Research Tools

Description

Fezf1-2A-dCre-D mice express a trimethoprim-inducible Cre recombinase directed to Fezf1-expressing cells by the endogenous promoter/enhancer elements of the Fez family zinc finger 1 locus. When induced, small increases in Cre recombinase activity are observed in restricted populations within striatum and hypothalamus, as well as scattered positive cells in amygdala and cortex.

Detailed description

The Fezf1-2A-dCre-D targeted mutation (also called Fezf1-2A-dCre-Δhygro) has a viral 2A oligopeptide sequence (T2A) that mediates ribosomal skipping and a destabilized Cre fusion gene (dCre) inserted downstream of the Fez family zinc finger 1 coding region. This is designed to have both endogenous gene and dCre expression directed to Fezf1-expressing cells by the endogenous promoter/enhancer elements.

The ecDHFR^R12Y/Y100I domain of dCre leads to proteosomal degradation of the entire fusion protein, resulting in reduced overall Cre recombinase activity. Administration of the DHFR inhibitor, trimethoprim (TMP), prevents degradation of the dCre fusion gene and results in small increases in Cre recombinase activity.

When Fezf1-2A-dCre-D mice are bred with mice containing loxP-flanked sequences, TMP-enhanced Cre recombination will result in deletion of floxed sequences in the Fezf1-expressing cells of the double mutant offspring. Specifically, the donating investigator reports that Fezf1-2A-dCre-D mice have trimethoprim-inducible increases in Cre recombinase activity (reporter gene expression) detected in restricted populations within striatum and hypothalamus, as well as scattered positive cells in amygdala and cortex, in a pattern that correlates well with endogenous Fezf1 expression. In the absence of trimethoprim, small reductions in Cre recombinase activity are observed in these brain regions. Heterozygous mice are viable and fertile with no gross physical or behavioral abnormalities. The donating investigator did not examine dCre activity in tissues other than brain, and did not attempt to generate homozygous mice to date (September 2014).

For characterization information, see images at the Allen Institute for Brain Science website (Fezf1-2A-dCre images).

The dCre fusion gene (also called destabilized Cre, hDHFR/Cre or ecDHFR^R12Y/Y100I/Cre) is Cre recombinase fused at its N terminus to the first 159 amino acids of the Escherichia coli K-12 strain chromosomal dihydrofolate reductase gene (DHFR or folA) harboring the G67S mutation and modified to also include the R12Y/Y100I destabilizing domain mutations. The ecDHFR^R12Y/Y100I domain of dCre leads to proteosomal degradation of the entire fusion protein, resulting in little or no Cre recombinase activity. The donating investigator reports that administration of the high affinity DHFR inhibitor trimethoprim (TMP; at a concentration of 0.25-0.30 mg/g body weight) prevents degradation of the dCre fusion gene, resulting in Cre recombinase activity.

Development

The Fezf1-2A-dCre-D targeted mutation (also called Fezf1-2A-dCre-Δhygro) has a viral 2A oligopeptide sequence (T2A) that mediates ribosomal skipping and a destabilized Cre fusion gene (dCre) inserted downstream of the Fez family zinc finger 1 coding region. The specific details are below.

(129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem (ES) cells, already targeted with a T2A-Cre vector immediately downstream of the Fez family zinc finger 1 translational STOP codon, were re-targeted with a "T2A-DHFR/Cre" vector and a FLP-expressing plasmid to facilitate recombination.

Correctly re-targeted ES cells had (from 5' to 3') a partial Fezf1 intron 3 sequence containing an frt3 site, a partial Fezf1 exon 4 sequence up to (but not including) the endogenous stop codon, a T2A cleavage sequence that is in-frame with the Fezf1 coding sequence, a dCre fusion gene (described below) that is in-frame with the Fezf1 coding sequence, a bovine growth hormone polyA sequence, an AttB sequence, a PGK-hygromycin-SV40polyA cassette (with an mRNA splice donor-frt5 site-mRNA splice acceptor in the hygromycin gene), and an AttP sequence.

The dCre fusion gene (also called destabilized Cre, DHFR/Cre or ecDHFR^R12Y/Y100I/Cre) is Cre recombinase fused at its N terminus to the first 159 amino acids of the Escherichia coli K-12 strain chromosomal dihydrofolate reductase gene (DHFR or folA) harboring the G67S mutation and modified to also include the R12Y/Y100I destabilizing domain mutations.

Correctly re-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-5'hygro::mRNA splice donor::frt5::mRNA splice acceptor::3'hygro::SV40 polyA) and replace it with the recombined AttB/AttP site (AttL). The resulting Fezf1-2A-dCre-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 2014. Upon arrival, males were used to cryopreserve sperm. To establish the living Fezf1-2A-dCre-D mouse colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).

Allele

Symbol: Fezf1^{tm1.1(cre/folA)Hze}
Name: targeted mutation 1.1, Hongkui Zeng
MGI accession ID: MGI:5603434
Generation method: Targeted
Attributes: Recombinase-expressing; Inducible
Marker symbol: Fezf1
Marker name: Fez family zinc finger 1
Chromosome: 6
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: cre/folA,cre/folA,
Site of expression: Upon induction, Cre recombinase activity is observed in restricted populations within striatum and hypothalamus, as well as scattered positive cells in amygdala and cortex.
Molecular note: ES cells previously targeted with a T2A-Cre vector immediately downstream of the Fez family zinc finger 1 translational STOP codon were re-targeted with a "T2A-hDHFR/Cre" vector and a FLP-expressing plasmid to facilitate recombination. Correctly re-targeted ES cells had (from 5' to 3') a partial Fezf1 intron 3 sequence containing an frt3 site, a partial Fezf1 exon 4 sequence up to (but not including) the endogenous stop codon, a T2A cleavage sequence that is in-frame with the Fezf1 coding sequence, a dCre fusion gene (described below) that is in-frame with the Fezf1 coding sequence, a bovine growth hormone polyA sequence, an AttB sequence, a PGK-hygromycin-SV40polyA cassette (with an mRNA spice donor-frt5 site-mRNA spice acceptor in the hygromycin gene), and an AttP sequence. The dCre fusion gene (also called destabilized Cre, DHFR/Cre or ecDHFR/Cre) is Cre recombinase fused at its N terminus to the first 159 amino acids of the Escherichia coli K-12 strain chromosomal dihydrofolate reductase gene (DHFR or folA) harboring the G67S mutation and modified to also include the R12Y/Y100I destabilizing domain mutations. PhiC31-mediation recombination removed the AttB/AttP-flanked sequence and replaced it with the recombined AttB/AttP site (AttL).