B6.Cg-Gt(ROSA)26Sor^{tm1(Stat3*A661C*N663C)Sbkv}/J

Stock number: 038243
Product name: R26STAT3C^stopfl
Research areas: Immunology, Inflammation and Autoimmunity Research, Research Tools

Description

R26STAT3C^stopfl knock-in mice express a Cre-inducible FLAG-tagged constitutively active Stat3C gene with a downstream IRES-EGFP sequence. Cre-mediated recombination results in the excision of a floxed STOP/neo sequence, enabling the tissue and time-dependent expression of bicistronic Stat3C and EGFP. This strain is useful when studying STAT3 hyperactivity during T lymphocyte differentiation.

Detailed description

R26STAT3C^stopfl mice contain a Gt(ROSA)26Sor knock-in allele containing, 5' to 3', a floxed stop/neo cassette, a hyperactive FLAG-tagged signal transducer and activator of transcription 3 (Stat3) cDNA (called Stat3C), and a frt-flanked internal ribosome entry site (IRES)-EGFP sequence. Stat3 encodes a transcription activator induced by cytokines and growth factors that acts as a regulator of inflammatory response by regulating differentiation of naive CD4(+) T-cells into T-helper Th17 or regulatory T-cells (Treg). STAT3 hyperactivity in T lymphocytes causes an expansion of Th17 cells, which home preferentially to the lungs and leads to chronic inflammation, increased mucus production, and decreased lung function. STAT3C differs from wild-type STAT3 in two cysteine substitutions, A661C and N663C, resulting in disulfide linkage between two STAT3 monomers brought into proximity by Y705 phosphorylation and dimer stabilization.

The targeted R26STAT3C^stopfl allele has an IRES-GFP sequence that leads to transcription of a bicistronic Stat3C EGFP mRNA, allowing for tracking of the cells in which the loxP-flanked stop cassette is deleted and STAT3C is expressed. When bred to CD4-cre mice (Stock No. 022071), FACS analysis shows nearly all peripheral T cells in the mutant mice express GFP.

Homozygous R26STAT3C^stopflfl mice are viable and fertile. The GFP fluorophore is detectable by direct fluorescence, immunostaining, and FACS analysis.

Development

The R26STAT3C^stopfl targeting vector was designed with, from 5' to 3', a floxed stop/neo cassette, a hyperactive FLAG-tagged signal transducer and activator of transcription 3 (Stat3) cDNA (called Stat3C), and a frt-flanked internal ribosome entry site (IRES)-EGFP sequence. STAT3C differs from wild-type STAT3 in two cysteine substitutions, A661C and N663C, resulting in disulfide linkage between two STAT3 monomers brought into proximity by Y705 phosphorylation and dimer stabilization. Final targeting vector, with 5' and 3' arms of homology in the Gt(ROSA)26Sor gene (between exons 1 and 2), was used to generate a conditional STAT3C allele by exon trapping. The construct was electroporated into B6.Cg-Thy1^a-derived Bruce4 embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts and resulting chimeric mice were bred to C57BL/6J mice (Stock No. 000664) for at least 3 generations. Upon arrival at The Jackson Laboratory, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes.

Allele

Symbol: Gt(ROSA)26Sor^{tm1(Stat3*A661C*N663C)Sbkv}
Name: targeted mutation 1, Sergei B Koralov
MGI accession ID: MGI:3778925
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Constitutively active; Inserted expressed sequence
Synonyms: Gt(ROSA)26Sor^{tm1(Stat3*)Sbkv}; Gt(ROSA)26Sor^{tm1(STAT3C)Sbkv}; Gt(ROSA)26Sor^{tm2(STAT3,EGFP)Cgn}; R26Stat3C^stopfl; Stat3-C^stoplox
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Marker synonyms: beta geo; Gtrgeo26; Gtrosa26; R26; ROSA26; SETD5-AS1; Thumpd3as1
Chromosome: 6
Strain of origin: B6.Cg-Thy1^a
Molecular note: A targeting construct introduced into the locus a floxed neomycin resistance and STOP cassette, the cDNA for a hyperactive version of human Stat3 (A661C and N663C; STAT3C) with a FLAG-tag, and an FRT-flanked IRES-EGFP reporter cassette. In the presence of Cre recombinase, the STOP element is removed and bicistronic expression of STAT3 and GFP occurs.