B6.129S-Rag2^tm1Fwa Cd47^tm1Fpl Il2rg^tm1Wjl/J

Stock number: 025730
Product name: Triple KO (TKO)
Research areas: Cancer Research, Immunology, Inflammation and Autoimmunity Research

Description

Triple Knockout mice lack RAG2, CD47, and X-linked IL2RG - making them tolerant of xenotransplantation, resistant to graft versus host disease, and susceptible to HIV.

Of note, a similar strain containing a RAG1 KO, instead of a RAG2 KO, is available as Stock No. 032088.

Detailed description

Triple knockout mice do not express the recombination activating gene 2 (Rag2), the CD47 antigen (CD47) gene, or the X-linked interleukin 2 receptor, gamma chain (Il2rg) gene. These mice are viable and fertile. These mice contain no T cells, B cell, or Natural Killer (NK) cells. The disruption of the Il2rg gene (γc) prevents the development of thymomas and delays the onset of graft versus host disease (GVHD). Recognition of human CD47 by the murine signal-regulatory protein alpha (SIRPα) is required to suppress macrophage-mediated phagocytosis of transplanted human hematopoietic stem cells (HSCs). C57BL/6 mice express a form of the SIRPα receptor that does not recognize human CD47, leading to a lack of tolerization of xenotransplantation. Since phagocytes developed in a CD47-negative environment are tolerized to cells that do not express CD47, disruption of murine CD47 in this C57BL/6 model negates the requirement for CD47-SIRPα signaling and induces tolerance to transplanted human cells.

After transplantation of human bone marrow, liver, and thymus into a similar model, mice develop organized lymphoid tissues and have an intact complement system. Transplantation results in a majority of leukocytes in the peripheral circulation being from human origin. These mice may be good in applications associated with graft versus host disease (GVHD) and virus-specific immune responses.

Development

A targeting vector was designed to replace 0.85kb of the recombination activating gene 2 (Rag2) with a neomycin resistance (neo) cassette and a polyadenylation (polyA) sequence. The construct was electroporated into 129S/SvEv-Gpi1^c-derived CCE embryonic stem (ES) cells. Correctly targeted ES cells were injected into C57BL/6 blastocysts and the resulting chimeric males were bred to females. Offspring were subsequently bred to mice bearing the X-linked Il2rg^tm1Wjl/J allele (Stock No. 003174). These mice lack exons 4-8 and part of exon 3 of the interleukin 2 receptor, gamma chain (γc). Rag2^-/-γc^-/- females were bred to B6.129S7-CD47^tm1Fpl/J (Stock No. 003173) males. Resulting Rag2^+/-γc^-/YCD47^+/- males were backcrossed to Rag2^-/-γc^-/-, and mating of F2 Rag2^-/-γc^-/-CD47^+/- females and Rag2^-/-γc^-/YCD47^+/- males produced this Rag2^-/-γc^-/-CD47^-/- (triple knockout; TKO) colony. These mice were bred to C57BL/6J mice (Stock No. 000664) for at least 10 generations.

Allele

Symbol: Rag2^tm1Fwa
Name: targeted mutation 1, Frederick W Alt
MGI accession ID: MGI:1858556
Generation method: Targeted
Attributes: Null/Knockout
Marker symbol: Rag2
Marker name: recombination activating gene 2
Chromosome: 2
Strain of origin: 129S/SvEv-Gpi1^c
Molecular note: A neomycin resistance cassette replaced a 0.85kb segment of the open reading frame, eliminating the encoding of 286 of the 527 amino acids of the protein.

Allele

Symbol: Il2rg^tm1Wjl
Name: targeted mutation 1, Warren J Leonard
MGI accession ID: MGI:1857455
Generation method: Targeted
Attributes: Null/Knockout
Marker symbol: Il2rg
Marker name: interleukin 2 receptor, gamma chain
Chromosome: X
Strain of origin: 129S4/SvJae
Site of expression: Primarily lymphoid cells.
Molecular note: A neomycin resistance cassette replaced part of exon 3 and all of exons 4 - 8 of the gene, resulting in the loss of most of the extracellular domain and all of the transmembrane and cytoplasmic domains of the protein.

Allele

Symbol: Cd47^tm1Fpl
Name: targeted mutation 1, Frederik P Lindberg
MGI accession ID: MGI:1861955
Generation method: Targeted
Attributes: Null/Knockout
Marker symbol: Cd47
Marker name: CD47 antigen (Rh-related antigen, integrin-associated signal transducer)
Chromosome: 16
Strain of origin: 129S7/SvEvBrd-Hprt^b-m2
Molecular note: A neomycin resistance cassette was inserted into exon 2 of the gene, which encodes the signal peptide cleavage site and the IgV domain. Monoclonal antibodies recognizing the Ig domain failed to detect any CD47 antigen on the surface of erythrocytes, bone marrow cells, or other cells including peripheral blood leukocytes, splenocytes, and fibroblasts from homozygous mutant mice. In addition, no antigen was detected in mutant erythrocyte lysates with polyclonal antisera to the NH2 or COOH-termini.