B6.129P2(Cg)-Rorc^tm2Litt/J

Stock number: 007572
Product name: Rorc(γt)-EGFP
Research areas: Apoptosis Research, Cancer Research, Developmental Biology Research, Immunology, Inflammation and Autoimmunity Research, Internal/Organ Research, Research Tools

Description

These mice harbor the reporter Rorcγt^GFP mutant and may be useful in studying immune system homeostasis, T cell repertoire selection, CD4/CD8 double positive thymocyte survival, lymphoid organogenesis, proinflammatory T-helper cell development, mucosal immunology, and the role of inflammatory disease in autoimmunity and cancer progression.

Detailed description

Mice homozygous for this Rorc(γt^GFP (or RORγt)^GFP) mutant allele are viable and fertile. While Rorcγ mRNA is detected in liver in Rorc(γ)t^GFP homozygotes, mRNA and protein for the thymus-specific isoform (Rorcγt) encoded by the targeted allele are not detected in the thymus. EGFP expression reports Rorc(γt) transcription in the thymi of adult Rorc(γt)GFP mice. Homozygous mice exhibit abnormal lymph node, Peyer's patch, and lymphoid tissue inducer (LTi) cell development. Mice with Rorcγt-deficient T cells lack tissue-infiltrating proinflammatory T-helper cells (Th17 cells), and are protected from induced autoimmune disease (EAE) on this genetic background.

The donating investigator also reports increased thymoma incidence in homozygotes, with 80-90% of homozygotes succumbing to thymomas by 12-14 weeks of age. It should be noted that the time to onset may vary due to vivarium-specific environmental factors. In the JAX Repository colony, we have observed that female homozygotes live to 9-11 weeks of age and most male homozygotes die before 8 months of age (October 2015). Some researchers observe a much earlier onset. These RorcγtGFP mutant mice may be useful in applications related to immune homeostasis, T cell repertoire selection, CD4/CD8 double positive (CD4⁺/CD8⁺) thymocyte survival, and the role of inflammatory disease in autoimmunity and cancer progression.

Development

A targeting vector was designed to insert the coding sequence of Enhanced Green Fluorescent Protein (EGFP), followed by a loxP-flanked neomycin resistance cassette, into "exon 1γt" directly downstream of the ATG translational start codon of the targeted gene. The construct was electroporated into the 129P2/OlaHsd-derived E14.1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts, and chimeras were bred to unspecified mice to generate Rorc(γt)^GFPneo heterozygotes. These mice were then crossed with a Cre-deleter strain (unspecified genetic background) to excise the neo cassette. The donating investigator reported that the resulting Rorc(γt)^GFP pups were then backcrossed to C57BL/6 (see SNP note below) for at least five generations prior to arrival at The Jackson Laboratory.

A 32 SNP (single nucleotide polymorphism) panel analysis, with 27 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the rederived living colony at The Jackson Laboratory Repository. While the 27 markers throughout the genome suggested a C57BL/6 genetic background, at least 1 of 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6J ; C57BL/6N genetic background.

Allele

Symbol: Rorc^tm2Litt
Name: targeted mutation 2, Dan R Littman
MGI accession ID: MGI:3026635
Generation method: Targeted
Attributes: Reporter; Null/Knockout
Marker symbol: Rorc
Marker name: RAR-related orphan receptor gamma
Chromosome: 3
Strain of origin: 129P2/OlaHsd
Expressed genes: GFP,Green Fluorescent Protein,
Site of expression: EGFP expression is reported in the thymus (cortex) of adult mice. EGFP is expressed in double-positive thymocytes, with small amounts of EGFP in CD4⁺ and CD8⁺ single-positive thymocytes.
Molecular note: An isoform-specific disruption was generated by inserting a cassette containing eGFP downstream of the start codon of exon 1gammat. The targeted exon is the third exon of the gene, serves as an alternative first exon for the gammat transcript, and is not included in the gamma transcript. RT-PCR analysis of hepatic RNA obtained from homozygous mutant mice identified gamma transcript but not the targted gammat transcript. The eGFP sequence included a stop codon but no splice sites or polyadenylation signals. A floxed neo cassette included in the targeting vector was excised from the germline via cre-mediated recombination.