Overview

Also Known As:CTLA-4/CD28 double KO

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.

Donating Investigator

Arlene H Sharpe, Harvard Medical School

Genetic overview

Genetic Background	Generation

Ctla4^tm1Shr

Allele Type	Gene Symbol	Gene Name
Targeted (Null/Knockout)	Ctla4	cytotoxic T-lymphocyte-associated protein 4

Cd28^tm1Shr

Allele Type	Gene Symbol	Gene Name
Targeted (Null/Knockout)	Cd28	CD28 antigen

View Genetics

Research Applications

Cancer Research
Immunology, Inflammation and Autoimmunity Research
Virology Research
Research Tools
Cell Biology Research
Internal/Organ Research
Hematological Research

View All Research Applications

Base Price

Starting at:

$2,854.50 Domestic price Cryo Recovery

View Price List

Details

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.

Control Suggestions

Approximate Controls

Additional Information

Considerations for Choosing Controls

Selected References

Beaton S; Cleary A; ten Have J; Bradley MP. 1994. Cloning and characterization of a fox sperm protein FSA-1. Reprod Fertil Dev 6(6):761-70PubMed: 7624517 MGI: J:25259
Mandelbrot DA; Oosterwegel MA; Shimizu K; Yamada A; Freeman GJ; Mitchell RN; Sayegh MH; Sharpe AH. 2001. B7-dependent T-cell costimulation in mice lacking CD28 and CTLA4. J Clin Invest 107(7):881-7PubMed: 11285307 MGI: J:68642
Tivol EA; Borriello F; Schweitzer AN; Lynch WP; Bluestone JA; Sharpe AH. 1995. Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity 3(5):541-7PubMed: 7584144 MGI: J:30393

View All References

Genetics

Ctla4^tm1Shr

Allele Symbol: Ctla4^tm1Shr

Allele Name	targeted mutation 1, Arlene H Sharpe
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.

Cd28^tm1Shr

Allele Symbol: Cd28^tm1Shr

Allele Name	targeted mutation 1, Arlene H Sharpe
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~~tm1Shr~~.

Disease/Phenotype

Disease Terms

Research Areas By Phenotype

This mouse can be used to support research in many areas including:

Cancer Research
- Genes Regulating Growth and Proliferation
- Other
- Growth Factors/Receptors/Cytokines
Immunology, Inflammation and Autoimmunity Research
- Autoimmunity
  - collagen induced arthritis
- Immunodeficiency
  - T cell deficiency
  - specific T cell deficiency
  - B and T cell deficiency
- Intracellular Signaling Molecules
- T Cell Receptor Signaling Defects
- Growth Factors/Receptors/Cytokines
- Inflammation
  - B and T cell deficiency
- Lymphoid Tissue Defects
  - B and T cell deficiency
Virology Research
- B and T Cell Deficiency
Research Tools
- Cell Biology Research
- Cancer Research
  - T cell deficiency
  - specific T cell deficiency
- Immunology, Inflammation and Autoimmunity Research
  - T Cell Receptor Deficiency
  - T cell deficiency
  - specific T cell deficiency
Cell Biology Research
- Transcriptional Regulation
- Genes Regulating Growth and Proliferation
- Cell Cycle Regulation
- Signal Transduction
Internal/Organ Research
- Lymphoid Tissue Defects
  - T cell deficiency
- Thymus Defects
  - B and T cell deficient
Hematological Research
- Immunological Defects
  - B and T cell deficiency

Mammalian Phenotype Terms by Genotype

The following phenotype information is associated with a similar, but not exact match to this JAX^® Mice strain

Genotype: Cd28^tm1Shr/Cd28^tm1Shr Ctla4^tm1Shr/Ctla4^tm1Shr
C.129S4-Cd28 Ctla4

cellular phenotype

decreased T cell proliferation
- in vitro T cell proliferation is reduced relative to wild-type cells

hematopoietic system phenotype

decreased CD4-positive, alpha beta T cell number
- Normal - however, T and B cell development is normal
- the number of CD4+ CD69+ T cells is reduced by 50% compared to in wild-type mice

decreased T cell proliferation
- in vitro T cell proliferation is reduced relative to wild-type cells

homeostasis/metabolism phenotype

abnormal interferon level
- upon secondary stimulation in vitro, interferon-gamma levels are increased relative to in wild-type cells

immune system phenotype

abnormal interferon level
- upon secondary stimulation in vitro, interferon-gamma levels are increased relative to in wild-type cells

abnormal interleukin secretion
- upon primary stimulation in vitro, IL-2 production is decreased by 50% relative to in wild-type cells
- upon secondary stimulation in vitro, IL-4 and IL-10 production is decreased relative to in wild-type cells

decreased CD4-positive, alpha beta T cell number
- Normal - however, T and B cell development is normal
- the number of CD4+ CD69+ T cells is reduced by 50% compared to in wild-type mice

decreased T cell proliferation
- in vitro T cell proliferation is reduced relative to wild-type cells

increased length of allograft survival
- allograft survival is increased compared to in wild-type mice but mice eventually reject grafts

Genotype: Cd28^tm1Shr/Cd28^tm1Shr Ctla4^tm1Shr/Ctla4⁺
C.129S4-Cd28 Ctla4

hematopoietic system phenotype

abnormal CD8-positive, alpha beta T cell morphology
- abnormally high levels of CD5 (a marker of T cell activation) and CD8 expression on T cells

immune system phenotype

abnormal CD8-positive, alpha beta T cell morphology
- abnormally high levels of CD5 (a marker of T cell activation) and CD8 expression on T cells

increased susceptibility to autoimmune disorder

References

Beaton S; Cleary A; ten Have J; Bradley MP. 1994. Cloning and characterization of a fox sperm protein FSA-1. Reprod Fertil Dev 6(6):761-70PubMed: 7624517 MGI: J:25259
Mandelbrot DA; Oosterwegel MA; Shimizu K; Yamada A; Freeman GJ; Mitchell RN; Sayegh MH; Sharpe AH. 2001. B7-dependent T-cell costimulation in mice lacking CD28 and CTLA4. J Clin Invest 107(7):881-7PubMed: 11285307 MGI: J:68642
Tivol EA; Borriello F; Schweitzer AN; Lynch WP; Bluestone JA; Sharpe AH. 1995. Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity 3(5):541-7PubMed: 7584144 MGI: J:30393

Additional - Cd28^tm1Shr related

Additional - Ctla4^tm1Shr related

Technical Support

CONTACT TECHNICAL SUPPORT

Genotyping Protocols
- Separated PCR:Ctla4
- Standard PCR:Cd28
- Genotyping resources and troubleshooting
Breeding Considerations

When maintaining a live colony, mice homozygous for both targeted mutations may be bred together.
- Additional Breeding and Husbandry Support
Citation
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.

Animal Health Reports

Facility Barrier Level Descriptions

Production of mice from cryopreserved embryos or sperm occurs in a maximum barrier room, G200

Pricing & Availability

Cryo Recovery

Domestic
International

Live Mouse

Age

Genotype

Price

weeks

Cryorecovery - Pricing

Service/Product	Description	Price
	Heterozygous for Cd28<tm1Shr>, Heterozygous for Ctla4<tm1Shr>

Related Products and Services

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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General Terms and Conditions

Questions about Terms of Use

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Licensing Information

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Also Known As:CTLA-4/CD28 double KO

Donating Investigator

Genetic overview

Research Applications

Base Price

Detailed Description

Development

Control Suggestions

Approximate Controls

Additional Information

Selected References

Ctla4tm1Shr

Allele Symbol: Ctla4tm1Shr

Cd28tm1Shr

Allele Symbol: Cd28tm1Shr

Disease Terms

Research Areas By Phenotype

This mouse can be used to support research in many areas including:

Mammalian Phenotype Terms by Genotype

Genotype: Cd28tm1Shr/Cd28tm1Shr Ctla4tm1Shr/Ctla4tm1ShrC.129S4-Cd28 Ctla4

cellular phenotype

hematopoietic system phenotype

homeostasis/metabolism phenotype

immune system phenotype

Genotype: Cd28tm1Shr/Cd28tm1Shr Ctla4tm1Shr/Ctla4+C.129S4-Cd28 Ctla4

hematopoietic system phenotype

immune system phenotype

References

Additional - Cd28tm1Shr related

Additional - Ctla4tm1Shr related

Genotyping Protocols

Breeding Considerations

Citation

Animal Health Reports

Production of mice from cryopreserved embryos or sperm occurs in a maximum barrier room, G200

Live Mouse

Cryorecovery - Pricing

Related Products and Services

Payment Terms and Conditions

The Jackson Laboratory's Genotype Promise

Terms Of Use

Additional Use Restrictions Apply

Licensing Information

JAX® Mice, Products & Services Conditions of Use

No Warranty

Credit for PRODUCTS or SERVICES

Animal Care and Use for SERVICES

No Liability

Ctla4^tm1Shr

Allele Symbol: Ctla4^tm1Shr

Cd28^tm1Shr

Allele Symbol: Cd28^tm1Shr

Genotype: Cd28^tm1Shr/Cd28^tm1Shr Ctla4^tm1Shr/Ctla4^tm1Shr
C.129S4-Cd28 Ctla4

Genotype: Cd28^tm1Shr/Cd28^tm1Shr Ctla4^tm1Shr/Ctla4⁺
C.129S4-Cd28 Ctla4

Additional - Cd28^tm1Shr related

Additional - Ctla4^tm1Shr related