STOCK Fos^{tm2.1(icre/ERT2)Luo}/J

Stock number: 030323
Product name: Fos^2A-iCreER (TRAP2)
Research areas: Cancer Research, Neurobiology Research, Research Tools

Description

Fos^2A-iCreER knock-in (also called Fos^2A-iCreERT2 or "TRAP2") was designed to have expression of a tamoxifen-inducible, improved Cre recombinase (icre/ERT2) from the Fos promoter/enhancer elements - without disrupting endogenous Fos expression. Fos^2A-iCreER mice are a Cre-lox tool allowing inducible iCre recombination in Fos-expressing cells/tissues (including neurons activated by specific somatosensory, visual and auditory stimuli) or behavioral experiences such as enriched environment, fear conditioning or water deprivation. Immediate early genes (IEGs) are the connection between gene expression and a neuron's electrical/synaptic activity (which defines neuronal response properties).

Detailed description

The Fos^2A-iCreER knock-in allele (also called Fos^2A-iCreERT2 or "TRAP2") was designed to have expression of a tamoxifen-inducible, improved Cre recombinase (iCreER^T2) from the Fos promoter/enhancer elements - without disrupting endogenous Fos expression. The donating investigator reports this allele preserves endogenous Fos, including the highly conserved first intron and the 3' untranslated region critical for mRNA destabilization. [DeNardo et al. 2019 Nat Neurosci. 22:460]
iCreER^T2 fusion protein activity is inducible; observed following tamoxifen administration. When Fos^2A-iCreER mice are bred with mice containing lox-flanked sequences, tamoxifen-inducible iCre-mediated recombination will result in deletion of floxed sequences in the Fos-expressing cells of the offspring.

Specifically, the donating investigator reports that following tamoxifen induction, Cre recombinase activity is observed in active populations of neurons in the brain - in a pattern consistent with endogenous Fos expression. Before tamoxifen exposure, some Cre recombination/activity is reported in the olfactory bulb - but those baseline levels are significantly less than the tamoxifen-induced levels of Cre recombination/activity. The donating investigator did not examine iCreER^T2 expression in tissues other than brain.

Prior to tamoxifen exposure, heterozygous Fos^2A-iCreER mice are viable and fertile with no behavioral, neurological or any other spontaneous abnormalities. Additionally, the Fos^2A-iCreER colony at The Jackson Laboratory is viable and fertile as homozygotes.

The iCreER^T2 fusion protein used here consists of a codon-improved Cre recombinase fused to a G400V/M543A/L544A 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, iCre/ER^T2 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 Fos^2A-iCreER knock-in allele (called Fos^2A-iCreERT2 or "TRAP2") was created by the laboratory of Dr. Liqun Luo (Stanford University). First, a targeting vector was designed to insert a 2A::iCreER^T2::frt-neo-frt cassette into the translational stop site of the FBJ osteosarcoma oncogene locus (Fos) on chromosome 12. The viral 2A oligopeptide sequence mediates ribosomal skipping. The iCreER^T2 fusion gene used here has an improved/optimized variant of Cre recombinase (iCre) that is fused to a G400V/M543A/L544A triple mutation of the human estrogen receptor ligand binding domain (ER^T2). The iCre is improved with mammalian codon usage, removed putative cryptic splice sites, altered stop codon and reduced CpG content to limit the chances of epigenetic silencing in mammals. The targeting construct was electroporated into 129;FVB-derived embryonic stem (ES) cells, and correctly targeted ES cells were injected into recipient blastocysts. Chimeric animals were bred to CD1 mice and/or germline-active GFP-FlpO transgenic mice (mixed genetic background including CD1) to delete the frt-flanked neo cassette. The resulting Fos^2A-iCreER (TRAP2) mice were bred to C57BL/6NCrl mice for 2-3 generations (and the FlpO-expressing transgene was removed), and then bred to Ai14 mice (B6;129S6-Gt(ROSA)26Sor^{tm14(CAG-tdTomato)Hze}/J from Stock No. 007908). The Fos^2A-iCreER colony was bred to C57BL/6NCrl mice for at least one additional generation (and the Ai14 allele was removed) prior to sending Fos^2A-iCreER males with black coat color to The Jackson Laboratory Repository in 2017. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6NJ oocytes (Stock No. 005304).

Of note, the donating investigator reports that, at least once during backcrossing, a Fos^2A-iCreER female was bred to a C57BL/6N inbred male (thus the Y chromosome of the congenic strain is of C57BL/6N origin).

The Jackson Laboratory colony of this strain initially incorporated animals that were black or albino. While no phenotypic differences associated with coat color have been noted here by our colony managers, a decision was made to breed out the albino allele for uniformity (March, 2023).

Allele

Symbol: Fos^{tm2.1(icre/ERT2)Luo}
Name: targeted mutation 2.1, Liqun Luo
MGI accession ID: MGI:5910950
Generation method: Targeted
Attributes: Recombinase-expressing; Inducible
Synonyms: Fos^2A-iCreER; Fos^2A-iCreERT2; TRAP2
Marker symbol: Fos
Marker name: FBJ osteosarcoma oncogene
Marker synonyms: AP-1; cFos; C-FOS; D12Rfj1; p55
Chromosome: 12
Strain of origin: 129
Expressed genes: cre/ERT2,Cre recombinase and estrogen receptor 1 (human) fusion gene,
Site of expression: When these mice are bred with mice containing lox-flanked sequences, tamoxifen-inducible iCre-mediated recombination will result in deletion of floxed sequences in the Fos-expressing cells of the offspring.
Molecular note: A targeting vector was designed to insert a 2A::iCreER::frt-neo-frt cassette into the translational stop site of the FBJ osteosarcoma oncogene locus (Fos) on chromosome 12. The viral 2A oligopeptide sequence mediates ribosomal skipping. The iCreER fusion gene used here has an improved/optimized variant of Cre recombinase (iCre) that is fused to a G400V/M543A/L544A triple mutation of the human estrogen receptor ligand binding domain (ER). The targeting construct was electroporated into 129;FVB-derived embryonic stem (ES) cells, and correctly targeted ES cells were injected into recipient blastocysts. Chimeric animals were bred to CD1 mice and/or germline-active GFP-FlpO transgenic mice (mixed genetic background including CD1) to delete the frt-flanked neo cassette.