B6.129(Cg)-Arc^{tm1.1(cre/ERT2)Luo}/J

Stock number: 021881
Product name: Arc^CreER
Research areas: Neurobiology Research, Research Tools

Description

The Arc^CreER knock-in/knockout allele was designed to both abolish expression of the Arc immediate early gene and direct expression of the CreER^T2 fusion protein from the Arc promoter/enhancer elements. These mice are a Cre-lox tool that allows inducible Cre expression in Arc-expressing cells/tissues; including neurons activated by specific somatosensory, visual, and auditory stimuli. Immediate early genes (IEGs) are the connection between gene expression and a neuron's electrical/synaptic activity (which defines the neuron's response properties).

Detailed description

The Arc^CreER knock-in/knockout allele was designed to both abolish activity regulated cytoskeletal-associated protein (Arc) gene function and express CreER^T2 (CreERT2) fusion protein from the endogenous Arc promoter/enhancer elements. The donating investigator did not determine if endogenous gene mRNA/protein is expressed from the mutant allele. CreERT2 fusion protein activity is inducible; observed following tamoxifen administration. When Arc^CreER mice are bred with mice containing loxP-flanked sequences, tamoxifen-inducible Cre-mediated recombination will result in deletion of floxed sequences in the Arc-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 Arc expression. Some CreERT2 activity is observed in the brain prior to tamoxifen exposure (sub-populations of neurons in the brain, including in layer 6 neocortical cells and dentate gyrus granule cells, as well as sparser populations of other cell types). Additionally, a small number of offspring show Cre recombinase activity in the germline (both with or without tamoxifen). The donating investigator did not examine CreERT2 activity in tissues other than brain.

While other Arc null mutations are published with behavioral/neurological defects and embryonic lethality, the donating investigator reports that Arc^CreER homozygotes are viable and fertile with no behavioral/neurological defects grossly observed. More detailed behavioral/physiological characterization of homozygous mice has not been evaluated to date (April 2013).

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

A targeting vector was designed to insert a CreER^T2-SV40 polyA cassette followed by an FRT5-flanked pSV40-NeoR-pA cassette into the the translational start site of the activity regulated cytoskeletal-associated protein gene (Arc). The CreER^T2 fusion gene (Cre-ER^T2) is Cre recombinase fused to a G400V/M543A/L544A triple mutation of the human estrogen receptor ligand binding domain. The targeting event displaced the introns and coding regions and replaced the endogenous 3prime UTRs (which contribute to mRNA destabilization and to Arc mRNA dendritic trafficking) with an exogenous SV40 polyadenylation signal (to promote high-level expression). All sequences 5prime to the translational start site are retained. The construct was electroporated into (129X1/SvJ x 129S1/Sv)F1-Kitl⁺-derived R1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts. The resulting chimeric animals were bred to germline-active GFP-FlpO transgenic mice (mixed genetic background of CD1;FVB;C57BL/6) to delete the FRT5-flanked pSV40-NeoR-pA cassette. The resulting Arc^CreER (Arc^CreERT2) mice were bred with C57BL/6J wildtype mice for at least six generations (and the GFP-FlpO transgene was removed) prior to sending to The Jackson Laboratory Repository in 2013. Upon arrival, males were used to cryopreserve sperm. To establish the living Arc^CreER mouse colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).

Allele

Symbol: Arc^{tm1.1(cre/ERT2)Luo}
Name: targeted mutation 1.1, Liqun Luo
MGI accession ID: MGI:5488904
Generation method: Targeted
Attributes: Recombinase-expressing; Inducible
Marker symbol: Arc
Marker name: activity regulated cytoskeletal-associated protein
Chromosome: 15
Strain of origin: (129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Expressed genes: cre/ERT2,Cre recombinase and estrogen receptor 1 (human) fusion gene,
Site of expression: Following tamoxifen induction, Cre recombinase activity is observed in active populations of neurons in the brain in a pattern consistent with endogenous Arc expression.
Molecular note: A targeting vector was designed to insert a cre/ERT2-SV40 polyA cassette followed by an FRT5-flanked pSV40-NeoR-pA cassette into the the translational start site of the activity regulated cytoskeletal-associated protein gene (Arc). The targeting event displaced the introns and coding regions and replaced the endogenous 3' UTRs (which contribute to mRNA destabilization and to Arc mRNA dendritic trafficking) with an exogenous SV40 polyadenylation signal (to promote high-level expression). All sequences 5' to the translational start site are retained. The resulting chimeric animals were bred to germline-active GFP-FlpO transgenic mice to delete the FRT5-flanked pSV40-NeoR-pA cassette.