B6.Cg-Rag2^tm1Fwa Sgsh^mps3a Cd47^tm1Fpl Il2rg^tm1Wjl/JnolJ

Stock number: 039001
Product name: MPSIIIA-TKO
Research areas: Cancer Research, Cell Biology Research, Immunology, Inflammation and Autoimmunity Research, Internal/Organ Research, Mouse/Human Gene Homologs, Research Tools

Description

Removal of this mouse colony is imminent. If live mice are needed for your studies, it is advised that they be ordered immediately. After removal, the mice will be available from a cryorecovery.

MPSIIIA-TKO (Triple Knock-Out) mice carry a spontaneous Sgsh mutation as well as three knock-out mutations of the Rag2, Cd47 and Il2rg genes. These MPSIIIA-TKO mice may be useful for studying mucopolysaccharidosis type III (MPSIII) disease and relevant cellular therapies as these mice are tolerant of xenotransplantation and resistant to graft versus host disease.

Detailed description

The N-sulfoglucosamine sulfohydrolase (sulfamidase) gene, or Sgsh, codes for an enzyme involved in the lysosomal degradation of heparan sulfate. Mutations in this gene are associated with the lysosomal storage disease mucopolysaccaridosis IIIA (MPSIIIA), also known as Sanfilippo syndrome A. These MPSIIIA-TKO (Triple Knock-Out) mice were generated by crossing mice that carry the spontaneous Sgsh^mps3a mutant allele (Stock No. 003780) to TKO mice that are homozygous null for the recombination activating gene 2 (Rag2), CD47 antigen (Cd47) and X-linked interleukin 2 receptor, gamma chain (Il2rg) genes (Stock No. 025730).

MPSIIIA-TKO mice that are homozygous for the Rag2^tm1Fwa allele, heterozygous for the spontaneous Sgsh^mps3a mutant allele, homozygous for the Cd47^tm1Fpl allele, and homozygous/hemizygous for the X-linked Il2rg^tm1Wjl allele are viable and fertile. Mice that are homozygous for the Sgsh^mps3a mutant allele demonstrate a complete loss of SGSH activity in brain tissue deficit in motor performance beginning at 10 weeks of age. These homozygous MPSIIIA-TKO mice demonstrate a phenotype similar to the original mouse line carrying the spontaneous Sgsh^mps3a mutant allele (Stock No. 003780) and are expected to become moribund by 7-10 months of age; however, life-span of these MPSIIA-TKO mice has not been full evaluated by the donating laboratory.

As the MPSIIIA-TKO mice do not express the Rag2, CD47, or Il2rg genes, they do not produce functional T cells, B cells, 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) and loss of murine CD47 induces tolerance to transplanted human cells. These MPSIIIA-TKO mice are therefore tolerant of xenotransplantation and resistant to graft versus host disease, making them a useful model for studying potential human stem cell therapies targeting mucopolysaccharidosis type IIIA (MPSIIIA) disease.

Development

This MPSIIIA-TKO (Triple Knock-Out) mouse line was generated by crossing mice that carry the spontaneous Sgsh^mps3a allele (Stock No. 003780) on chromosome 11 to TKO mice that are homozygous null for the recombination activating gene 2 (Rag2) on chromosome 2, the CD47 antigen (Cd47) on chromosome 16 and X-linked interleukin 2 receptor, gamma chain (Il2rg) genes (Stock No. 025730).

The Sgsh^mps3a mutation arose in a colony developed from a chimera formed by injecting WW6 embryonic stem cells with a targeted mutation in the Mgat3 gene into C57BL/6 blastocysts. The ES cell clone did not carry the mutation. A female chimera was subsequently crossed with a CD1 male and Mgat3 heterozygotes were intercrossed. The Sgsh^mps3a mutation was detected after 4 generations and probably arose during intercrossing. The lysosomal storage disease phenotype segregated from the Mgat3^tm1Pst allele and a strain carrying the sulfamidase deficiency was developed. Thus, the genetic background was originally a mixture of 129SvJ, C57BL/6, SJL, and CD1. Thereafter, the mutation was backcrossed onto C57BL/6Jfor at least 14 generations and offered as Stock No. 003780.

To generate the TKO mouse line, 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 and offered as Stock No. 025730.

Dr. Jan Nolta purchased these two mouse strains from The Jackson Laboratory (Stock No. 003780 and Stock No. 027530) and intercrossed these lines for a minimum of 4 generations to produce the MPSIIA-TKO mouse line referred to as Stock No. 039001. Upon arrival at The Jackson Laboratory, embryos that were homozygous for the Rag2^tm1Fwa allele, heterozygous for the spontaneous Sgsh^mps3a allele, homozygous for the Cd47^tm1Fpl allele, and homozygous/hemizygous for the X-linked Il2rg^tm1Wjl allele were cryopreserved. These embryos were recovered and utilized to establish the live colony.

Allele

Symbol: Rag2^tm1Fwa
Name: targeted mutation 1, Frederick W Alt
MGI accession ID: MGI:1858556
Generation method: Targeted
Attributes: Null/Knockout
Synonyms: RAG-2-; Rag2-; RAG2 KO; Rag2^tm1Alt; Rag2^tm1Fwe; Rag2M
Marker symbol: Rag2
Marker name: recombination activating gene 2
Marker synonyms: Rag-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: Sgsh^mps3a
Name: mucopolysaccharidosis IIIA
MGI accession ID: MGI:2151181
Generation method: Spontaneous
Synonyms: MPS IIIA
Marker symbol: Sgsh
Marker name: N-sulfoglucosamine sulfohydrolase (sulfamidase)
Marker synonyms: 4632406A19Rik; HSS; MPS3A; SFMD; sulphamidase
Chromosome: 11
Strain of origin: Mixed stock
Molecular note: A G-to-A transition point mutation at coding nucleotide 91 of the encoded mRNA (c.91G>A) altered the corresponding amino acid from aspartic acid to asparagine at position 31 of the encoded protein (p.D31N).

Allele

Symbol: Il2rg^tm1Wjl
Name: targeted mutation 1, Warren J Leonard
MGI accession ID: MGI:1857455
Generation method: Targeted
Attributes: Null/Knockout
Synonyms: [KO]gammac; CD132^-; gammac^-; gc^-; Il2rg^tm1Wjll; IL2Rgamma^null
Marker symbol: Il2rg
Marker name: interleukin 2 receptor, gamma chain
Marker synonyms: [g]c; Ab2-183; Cd132; CIDX; common cytokine receptor gamma chain; common gamma chain; gamma C receptor; gamma(c); gammaC; gc; IL-2RG; IMD4; P64; SCIDX; SCIDX1
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
Synonyms: CD47^-; IAP^-; Itgp^tm1Fpl
Marker symbol: Cd47
Marker name: CD47 antigen (Rh-related antigen, integrin-associated signal transducer)
Marker synonyms: 9130415E20Rik; B430305P08Rik; IAP; integrin-associated protein; Itgp; MER6; OA3
Chromosome: 16
Strain of origin: 129S7/SvEvBrd-Hprt1^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.