B6(Cg)-Foxp3^{tm1.1(flpo)Daav}/J

Stock number: 038534
Product name: Foxp3^{Ametrine-FlpO}
Research areas: Immunology, Inflammation and Autoimmunity Research, Research Tools

Description

Foxp3^{Ametrine-FlpO} knock-in mice are designed to have an Ametrine fluorescent reporter, and optimized Flp-recombinase (FlpO) expression directed to forkhead box P3 (Foxp3)-expressing cells. These mice may be useful for studying regulatory T cell proliferation, localization, and antigen independence during the primary immune response.

Detailed description

FOXP3 is a member of the forkhead transcription factor family and involved in regulatory T cell (T_reg) differentiation and function. These Foxp3^{Ametrine-FlpO} knock-in mice express an Ametrine reporter and an FlpO recombinase sequence under the direction of the forkhead box P3 (Foxp3) promoter/enhancer elements. The Ametrine reporter allows tracking of the regulatory T cells while the optimized Flp (FlpO) recombinase allows Treg cell-specific deletion of Frt-flanked genes.

Homozygous female and hemizygous male Foxp3^{Ametrine-FlpO} knock-in mice are viable and fertile with no overt phenotype. Ametrine expression can be measured by flow cytometry. When Foxp3^{Ametrine-FlpO} mice are bred with mice containing FRT-flanked sequences, FlpO-mediated recombination will result in deletion of the FRT-flanked sequences in Foxp3-expressing cells in the offspring.

The donating investigator bred these Foxp3^{Ametrine-FlpO} mice to ThPOK^{F/F.iCreERT2.hCD2} transgenic mice (Stock No. 038538), to evaluate T cell subpopulations. Of the resulting progeny, those that were negative for iCre/ERT2 but positive for FlpO recombinase expressed the Ametrine reporter on T_reg cells exclusively as expected. These Foxp3^{Ametrine-FlpO} knock-in mice may be useful for T cell lineage tracing.

Development

The Foxp3^{Ametrine-FlpO} knock-in allele was generated by inserting an Ametrine reporter and FlpO (a codon-optimized flippase) cDNA into the ATG start codon of the forkhead box P3 (Foxp3) gene. The targeting construct consists of an Ametrine reporter following by a 2A peptide linker sequence, an FlpO cDNA followed by a second 2A peptide linker sequence, an AttB-Neo-AttP selection cassette and the full-length Foxp3 gene. The construct was electroporated into C57BL/6N-A^tm1Brd-derived JM8A3.N1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into C57BL/6 blastocysts and the resulting chimeric mice were crossed to C57BL/6 for one generation to confirm germline transmission. Foxp3^{Ametrine-FlpO} heterozygous mice were then bred to Rosa.PhiC31 mice to remove the Neo cassette.

The donating laboratory then bred these Foxp3^{Ametrine-FlpO} mice, with Neo cassette removed, to ThPOK^{F/F.iCreERT2.hCD2} (Stock No. 038538) transgenic mice, on a C57BL/6 background, for more than 15 generations. Males hemizygous for the Foxp3^{Ametrine-FlpO} knock-in allele and wildtype/non-carrier for the ThPOK^{F/F.iCreERT2.hCD2} transgene were used to cryopreserve this mouse line. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

Allele

Symbol: Foxp3^{tm1.1(flpo)Daav}
Name: targeted mutation 1.1, Dario AA Vignali
MGI accession ID: MGI:7414284
Generation method: Targeted
Attributes: Recombinase-expressing; Reporter
Synonyms: Foxp3^{Ametrine-FlpO}
Marker symbol: Foxp3
Marker name: forkhead box P3
Marker synonyms: AIID; DIETER; IPEX; JM2; PIDX; RGD1562112; scurfin; XPID
Chromosome: X
Strain of origin: C57BL/6N-A^tm1Brd
Molecular note: An Ametrine-2A and FlpO (a codon-optimized flippase) cDNS followed by a 2A peptide linker was inserted after the ATG start codon of the Foxp3 gene with a AttB-Neo-AttP cassette. Germline mice were then crossed with Rosa.PhiC31 mice to remove the Neo cassette.