B6(Cg)-Tg(Zbtb7b/CD4-icre/ERT2,-CD2)13Daav/J

Stock number: 038538
Product name: ThPOK^{F/F.icreERT2.hCD2}
Research areas: Immunology, Inflammation and Autoimmunity Research, Research Tools

Description

ThPOK^{F/F.iCreERT2.hCD2} transgenic mice express an FRT-flanked tamoxifen-inducible Cre-recombinase under the control of the CD4 antigen (Cd4) enhancer and promoter and zinc finger and BTB domain containing 7B (Zbtb7b) silencer, which drives Cre activity in CD4+ cells. The transgenic construct also expresses human CD2 antigen. These mice may be useful for deletion of loxP-flanked sequences in CD4-expressing cells.

Detailed description

The zinc finger transcription factor ThPOK (encoded by Zbtb7b) promotes CD4+ T cell development, as ThPOK silences genes that promote the CD8+ T cell lineage. During thymocyte maturation, ThPOK is expressed after positive selection and increases in major histocompatibility complex (MHC) class II selected thymocytes, resulting in direction to the CD4+CD8- helper lineage. The Zbtb7b silencer, a cis-regulatory element, represses Zbtb7b gene expression in CD4+CD8+ double positive precursors during commitment to the CD8+ T cell lineage. ThPOK^{F/F.iCreERT2.hCD2} transgenic mice express an FRT-flanked tamoxifen-inducible Cre-recombinase under the control of the CD4 antigen (Cd4) enhancer/promoter and zinc finger and BTB domain containing 7B (Zbtb7b) silencer, which drives Cre activity in CD4+ cells. The transgenic construct also expresses human CD2 which can be detected by flow cytometry. The donating investigator confirmed that the transgene was hemizygously inserted into chromosome 13 at approximately 13D1 (Immunity, 2021).

Restricted to the cytoplasm, Cre-ERT can only gain access to the nuclear compartment after exposure to tamoxifen. When ThPOK^{F/F.iCreERT2.hCD2} transgenic mice are bred with mice containing loxP-flanked sequences, tamoxifen-inducible Cre-mediated recombination will result in deletion of the floxed sequences in the cre-expressing cells of the offspring. Alternatively, when the ThPOK^{F/F.iCreERT2.hCD2} mice are bred with mice expressing a FLP-recombinase, the FRT-flanked iCre/ERT2 cassette will be deleted.

Hemizygous ThPOK^{F/F.iCreERT2.hCD2} transgenic mice are viable and fertile, with no overt phenotype. The donating laboratory has not evaluated homozygous transgenic mice. These ThPOK^{F/F.iCreERT2.hCD2} transgenic mice were crossed with B6.Cg-Gt(ROSA)26Sor^{tm14(CAG-tdTomato)Hze}/J mice (Stock No. 007914) to evaluate Cre activity. tdTomato expression was measured on CD4+ T cells following tamoxifen administration, with approximately 90% of hCD2+ CD4+ T cells expressing tdTomato. The donating laboratory also bred these ThPOK^{F/F.iCreERT2.hCD2} transgenic mice to Foxp3^{Ametrine-FlpO} knock-in mice (Stock No. 038534), resulting in progeny referred to as CD4conv^{iCreERT2-hCD2}. The CD4conv^{iCreERT2-hCD2} mice expressed iCreERT2 expression exclusively in CD4+ Foxp3- conventional T cells (Tconv) but not in regulatory T cells (Treg). These mice may be useful for deletion of loxP-flanked sequences in CD4-expressing cells and for tracking T cell subpopulations.

Development

The ThPOK^{F/F.iCreERT2.hCD2} transgenic construct was designed by inserting a 562base pair fragment of the zinc finger and BTB domain containing 7B (Zbtb7b) silencer sequence immediately downstream of the CD4 antigen (Cd4) enhancer and promoter sequences, followed by an FRT site, and tamoxifen inducible codon improved Cre recombinase (iCreERT2)cDNA. The sequence encoding an internal ribosomal entry site (IRES) and human CD2 antigen (CD2) were also inserted downstream of the cDNA cassette encoding iCreERT2 as well as an additional FRT site after the STOP codon of the human CD2 sequence. The construct was linearized and microinjected into fertilized C57BL/6 oocytes. Founder line number 13 was assigned for the transgene, which was bred to C57BL/6 mice for at least one generation to confirm germline transmission. The donating laboratory then bred hemizygous ThPOK^{F/F.iCreERT2.hCD2} transgenic mice to Foxp3^{Ametrine-FlpO} mice (Stock No. 038534) for more than 15 generations for colony maintenance. Males hemizygous for the ThPOK^{F/F.iCreERT2.hCD2} transgene and wildtype for the Foxp3^{Ametrine-FlpO} knock-in allele were used to cryopreserve this mouse line. The donating laboratory has determined that the transgene inserted into chromosome 13 at approximately 13D1(Immunity, 2021). To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J (Stock No. 000664) oocytes.

Allele

Symbol: Tg(Zbtb7b/CD4-icre/ERT2,-CD2)13Daav
Name: transgene insertion 13, Dario AA Vignali
MGI accession ID: MGI:7414283
Generation method: Transgenic
Attributes: Conditional ready (e.g. floxed); Recombinase-expressing; Inducible; Epitope tag
Synonyms: ThPOK^{F/F.iCreERT2.hCD2}
Marker symbol: Tg(Zbtb7b/CD4-icre/ERT2,-CD2)13Daav
Marker name: transgene insertion 13, Dario AA Vignali
Chromosome: 13
Strain of origin: C57BL/6
Molecular note: A 562–base pair fragment of the Zbtb7b silencer sequence was inserted into a mini-transgene containing the minimal Cd4 enhancer-promoter-FRT-icre/ERT2. A FRT site was inserted 1086bp upstream of the ATG codon of the icre/ERT2. The sequence encoding an internal ribosomal entry site and human CD2 were also inserted downstream of the cDNA cassette encoding icre/ERT2 and additional FRT site was inseted after the STOP codon of the CD2. The transgene expresses Cre specifically in the CD4 lineage. Founder information was not available. Line number 13 was assigned for the transgene inserted into chromosome 13 at approximately 13D1.