B6.129S4-Cd28^tm1Shr Ctla4^tm1Shr/J

Stock number: 017607
Product name: CTLA-4/CD28 double KO
Research areas: Cancer Research, Cell Biology Research, Hematological Research, Immunology, Inflammation and Autoimmunity Research, Internal/Organ Research, Research Tools, Virology Research

Description

These mice harbor both the Cd28 and the Ctla4 knock-out mutations on the same heterologous chromosome. These double mutant mice may be useful for studying T cell responses, activation, tolerance, development, co-stimulatory molecules, and allograft survival.

Detailed description

These mice harbor both the Cd28 and the Ctla4 knockout mutations on the same heterologous chromosome. Mice homozygous for both knockout mutations (CD28/CTLA-4^-/-) are viable and fertile, with no lymphoproliferative phenotype. Flow cytometry shows CD28 and CTLA4 expression is abolished in homozygous mice (as determined by cell-surface CD28 staining of naive CD4⁺ T cells, and intracellular CTLA4 staining of activated CD4⁺ cells). CD28/CTLA-4^-/- mice congenic on the C57BL/6 genetic background (C57BL6.CD28/CTLA-4^-/-) exhibit decreased CD4⁺ T cells populations, decreased T cell proliferation, normal T cell / B cell development, and increased allograft survival (eventual heart graft rejection) when compared to wildtype mice. While mice singly homozygous for the CTLA-4 knockout mutation uniformly develop a lethal autoimmune lymphoproliferative disorder (death as early as 3 weeks of age) that is strictly dependent on CD28 costimulation, C57BL6.CD28/CTLA-4^-/- mice exhibit no lymphoproliferative phenotype (no splenomegaly, lymphadenopathy, or lymphocytic infiltrates), and their T cells are not in a constitutively activated state.

Development

A targeting vector was designed to replace exon 2 and a portion of exon 3 (collectively encoding IgV and part of a transmembrane domain) of the cytotoxic T-lymphocyte-associated protein 4 gene (Ctla4) on chromosome 1 with a PGK-neo cassette. This construct was electroporated into 129S4/SvJae-derived J1 embryonic stem (ES) cells. ES cell clone T5 was found to be heterozygous for the CTLA4 mutation and was able to gave rise to CTLA4-deficient mice exhibiting a lymphoproliferative phenotype. Because of the close proximity of Ctla4 and the CD28 antigen gene (Cd28) on chromosome 1, T5 ES cells were later re-targeted with a vector designed to replace part of the IgV-like exon 2 and a portion of transmembrane exon 3 of Cd28 with a PGK-hygro cassette. Correctly targeted ES cells bearing both mutations were injected into recipient blastocysts. The resulting chimeric animals were bred to C57BL/6J wildtype mice to generate the double mutant colony. Because of their close proximity, Ctla4 Cd28 double mutant mice were bred together by selecting progeny in which the two mutations co-segregated (indicating both mutations were on the same heterologous chromosome). Next, double mutant mice were backcrossed to C57BL/6 wildtype mice for at least ten generations (see SNP results below). In 2013, males homozygous for both knockout mutations (CD28/CTLA-4^-/-) were sent to The Jackson Laboratory Repository and used to cryopreserve sperm. To establish the living mouse colony, an aliquot of the frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

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 first generation rederived living colony at The Jackson Laboratory Repository. While the 27 markers throughout the genome suggested a C57BL/6 genetic background, 2 of 5 markers that determine C57BL/6J from C57BL/6N (one each on chromosome 8 and 19) were found to be heterozygous. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6J;C57BL/6N genetic background.

Allele

Symbol: Ctla4^tm1Shr
Name: targeted mutation 1, Arlene H Sharpe
MGI accession ID: MGI:2180668
Generation method: Targeted
Attributes: Null/Knockout
Marker symbol: Ctla4
Marker name: cytotoxic T-lymphocyte-associated protein 4
Chromosome: 1
Strain of origin: 129S4/SvJae
Molecular note: The insertion of a PGK-neo cassette replaced a 1.3 kb region containing exon 2 and a portion of exon 3. The deleted region encoded a IgV and part of a transmembrane domain. Fluorescence-activated cell sorting (FACS) analysis showed an absence of protein in splenocytes isolated from homozygous mutant mice.

Allele

Symbol: Cd28^tm1Shr
Name: targeted mutation 1, Arlene H Sharpe
MGI accession ID: MGI:3721239
Generation method: Targeted
Attributes: Null/Knockout
Marker symbol: Cd28
Marker name: CD28 antigen
Chromosome: 1
Strain of origin: 129S4/SvJae
Molecular note: A PGK-hygro cassette replaced a 3 kb region encompassing portions of exon 2 and exon 3. The deleted region encoded parts of a IgV-like domain and a transmembrane domain. Fluorescence-activated cell sorting (FACS) analysis showed an absence of protein on naive CD4 T cells isolated from the lymph nodes of homozygous mutant mice. Targeting was performed on an embryonic stem cell clone that was heterozygote for a targeted mutation of Ctla4tm1Shr.