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.
Arlene H Sharpe, Harvard Medical School
Genetic Background | Generation |
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|
Allele Type | Gene Symbol | Gene Name |
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Targeted (Null/Knockout) | Ctla4 | cytotoxic T-lymphocyte-associated protein 4 |
Allele Type | Gene Symbol | Gene Name |
---|---|---|
Targeted (Null/Knockout) | Cd28 | CD28 antigen |
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.
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 Name | targeted mutation 1, Arlene H Sharpe |
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Allele Type | Targeted (Null/Knockout) |
Allele Synonym(s) | CTLA-4-; Ctla4- |
Gene Symbol and Name | Ctla4, cytotoxic T-lymphocyte-associated protein 4 |
Gene Synonym(s) | |
Strain of Origin | 129S4/SvJae |
Chromosome | 1 |
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 Name | targeted mutation 1, Arlene H Sharpe |
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Allele Type | Targeted (Null/Knockout) |
Allele Synonym(s) | Cd28- |
Gene Symbol and Name | Cd28, CD28 antigen |
Gene Synonym(s) | |
Strain of Origin | 129S4/SvJae |
Chromosome | 1 |
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 Ctla4 |
When maintaining a live colony, mice homozygous for both targeted mutations may be bred together.
When using the CTLA-4/CD28 double KO mouse strain in a publication, please cite the originating article(s) and include JAX stock #017607 in your Materials and Methods section.
Facility Barrier Level Descriptions
Service/Product | Description | Price |
---|---|---|
Heterozygous for Cd28<tm1Shr>, Heterozygous for Ctla4<tm1Shr> |
Frozen Mouse Embryo | B6.129S4-Cd28<tm1Shr> Ctla4<tm1Shr>/J Frozen Embryo | $2595.00 |
Frozen Mouse Embryo | B6.129S4-Cd28<tm1Shr> Ctla4<tm1Shr>/J Frozen Embryo | $2595.00 |
Frozen Mouse Embryo | B6.129S4-Cd28<tm1Shr> Ctla4<tm1Shr>/J Frozen Embryo | $3373.50 |
Frozen Mouse Embryo | B6.129S4-Cd28<tm1Shr> Ctla4<tm1Shr>/J Frozen Embryo | $3373.50 |
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The Jackson Laboratory has rigorous genetic quality control and mutant gene genotyping programs to ensure the genetic background of JAX® Mice strains as well as the genotypes of strains with identified molecular mutations. JAX® Mice strains are only made available to researchers after meeting our standards. However, the phenotype of each strain may not be fully characterized and/or captured in the strain data sheets. Therefore, we cannot guarantee a strain's phenotype will meet all expectations. To ensure that JAX® Mice will meet the needs of individual research projects or when requesting a strain that is new to your research, we suggest ordering and performing tests on a small number of mice to determine suitability for your particular project. We do not guarantee breeding performance and therefore suggest that investigators order more than one breeding pair to avoid delays in their research.
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