NOD.Cg-Rag1^tm1Mom Il2rg^tm1Wjl Tg(HLA-DRA,HLA-DRB1*0401)39-2Kito/ScasJ

Stock number: 017914
Product name: DRAG
Research areas: Cancer Research, Hematological Research, Immunology, Inflammation and Autoimmunity Research, Internal/Organ Research, Research Tools, Virology Research

Description

DRAG mice are NOD.Rag1KO.IL2RγcKO ("NRG") animals with chimeric human-mouse class II transgenes encoding the HLA class II antigen binding domain molecules (defined by the HLA-DR4 genotype [HLA-DRA/HLA-DRB1*0401]) fused to the I-E^d MHC class II molecule. The presence of these HLA-DR4-IE transgenes allows enhanced HLA-DR-matched hematopoietic stem cells (HSC) engraftment and subsequent human T cell and B cell development. Because the human HLA-DR4 genotype is associated with the development of autoimmune diseases (rheumatoid arthritis, multiple sclerosis, type 1 diabetes, and lyme disease-induced arthritis), these DRAG mice may be useful as an in vivo model for studying human T cell/B cell development and function, vaccine testing, and generation of "fully human" IgM, IgG, IgA or IgE monoclonal antibodies for prophylactic and/or therapeutic use in autoimmune diseases and infectious diseases.

Detailed description

DRAG mice are NOD.Rag1KO.IL2RγcKO ("NRG"; Stock No. 007799) animals with chimeric human-mouse class II transgenes encoding the HLA class II antigen binding domain molecules (defined by the HLA-DR4 genotype [HLA-DRA/HLA-DRB1*0401]) fused to the I-E^d MHC class II molecule. The presence of these HLA-DR4-IE transgenes allows irradiated DRAG mice to be engrafted with HLA-DR-matched hematopoietic stem cells (HSC); resulting in humanized T cell and B cell populations.

DRAG females homozygous for both mutations and homozygous for the co-injected transgenes (and DRAG males homozygous for the Rag1KO mutation; hemizygous for the X-linked IL2RγcKO mutation; homozygous for the co-injected transgenes) are viable and fertile, with no mature T cells, B cells, or natural killer (NK) cells. Like NRG mice, DRAG mice have the same irradiation/engraftment advantages compared to NOD.scidKO.IL2RγcKO ("NSG"; Stock No. 005557), NOD.Rag1KO (Stock No. 003729), and NOD.scidKO (Stock No. 001303) mice. DRAG mice differ from NRG mice only in that DRAG mice have transgenic expression of HLA class II antigen-binding domain molecules (HLA-DR4) under the I-E^d promoter. In humans, the HLA-DR4 genotype (HLA-DRA/HLA-DRB1*0401) is associated with the development of autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, type 1 diabetes, and lyme disease-induced arthritis. The HLA-DR4-IE transgenes allow irradiated DRAG mice to be engrafted with HLA-DR-matched HSC; resulting in humanized T cell and B cell populations.

Specifically, when irradiated DRAG and NRG mice are engrafted with HLA-DR-matched umbilical cord blood HSC, the HLA-DR4-expression in DRAG mice results in several advantages compared to NRG mice:
i) DRAG mice show improved early engraftment and expansion of HLA-DR-matched hematopoietic stem cells in bone marrow.
ii) Such engraftment results in homing and development of bone-marrow-derived human T cell precursors in thymus, as well as proficient repopulation of peripheral lymphoid organs with mature CD4 cells and CD8 T cells.
iii) The repopulation of mature CD8 T cells in the periphery is noteworthy as DRAG mice do not express HLA class I molecules (the restriction element for thymic differentiation of human CD8 T cells).
iv) The T regulatory cell compartment (human CD4+FOXP3+ Tregs) is also reconstituted.
v) While engrafted DRAG and NRG mice develop similar human dendritic cell (spleen) and human B cell (periphery or spleen) percentages compared to human populations, the human B cells in DRAG mice are functional and secrete all human immunoglobulin classes (whereas human B cells in NRG mice secrete only IgM and have impaired class-switching).
vi) Neither DRAG or NRG mice develop significant populations of human natural killer (NK) cells in the spleen following engraftment.

Development

DRAG mice are NOD.Rag1KO.IL2RγcKO ("NRG") animals expressing HLA-DRB1*0401 molecules from the co-injected HLA-DR4-IE transgenes. DRAG mice harbor several mutations/transgenes, as described below.

Creation of the Rag1^null (Rag1KO or Rag1^tm1Mom) mutation on chromosome 2 is described for JAXMice Stock No. 003729. NOD-congenic mutant mice (NOD.Rag1KO mice) were used for breeding as below.

Creation of NOD congenic mice with the Rag1^null (Rag1KO or Rag1^tm1Mom) mutation on chromosome 2 and the IL2rγ^null (IL2RγcKO or Il2rg^tm1Wjl) mutation on the X chromosome is described for JAXMice Stock No. 007799. NOD-congenic double mutant mice (NOD.Rag1KO.IL2RγcKO or "NRG" mice) were used for breeding as below.

The MHC class II α chain is encoded by HLA-DRA, while the MHC class II β chain is encoded by different polymorphic alleles of HLA-DRB. In humans, the HLA-DR4 genotype (HLA-DRA / HLA-DRB1*0401) is associated with the development of autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, type 1 diabetes, and lyme disease-induced arthritis. Dr. Kouichi Ito (while at Hoffmann-La Roche Inc) generated mice possessing two HLA-DR4-IE chimeric MHC class II genes each encoded by its own HLA-DR4-IE transgene, HLA-DRA-IE^dα and HLA-DRBI*0401-IE^dβ. The HLA-DRA-IE^dα transgene has the mouse IE^dα genomic DNA sequences with its exon 2 replaced by the human HLA-DRA exon 2 sequence; the resulting chimeric human-mouse MHC class II gene encodes the IE^dα membrane-proximal domains with the antigen binding domain from HLA-DRA. The HLA-DRBI*0401-IE^dβ transgene has the mouse IE^dβ genomic DNA sequences with its exon 2 replaced by the human HLA-DRBI*0401 exon 2 sequence; the resulting chimeric human-mouse MHC class II gene encodes the IE^dβ membrane-proximal domains with the antigen binding domain from HLA-DRBI*0401. The two transgenes were co-injected into C57BL/6 fertilized eggs, and founder mice were obtained. Mice from founder line 39-2 were then backcrossed to C57BL/6-congenic MHC class II-deficient mice (H2-Ab1^tm1Gru). The resulting Abb Knockout/Transgenic HLA-DR4 mice were sent to Taconic Farms Inc (Taconic Model Number 4149; B6.129S2-H2-Ab1^tm1Gru Tg(HLA-DRA/H2-Ea,HLA-DRB1*0401/H2-Eb)1Kito). The mice were obtained for breeding as below.

To generate the DRAG animals, Dr. Sofia Casares (Naval Medical Research Center/Walter Reed Army Institute of Research) first obtained C57BL/6-congenic Abb Knockout/Transgenic HLA-DR4 mice (Taconic Model Number 4149) and backcrossed them 12 generations with NOD.Rag1KO mice (JAXMice Stock No. 003729). This also replaced the H2-Ab1^tm1Gru mutation with the endogenous H2-Ab1 allele. Next, mice were bred with "NRG" animals (JAXMice Stock No. 007799) to generate the DRAG mice. In 2012, DRAG mice were sent to The Jackson Laboratory Repository as JAXMice Stock No. 017914. Upon arrival, DRAG males were used to cryopreserve sperm. To establish the living DRAG mouse colony, an aliquot of the frozen sperm was used to fertilize oocytes from "NRG" females (JAXMice Stock No. 007799).

Allele

Symbol: Tg(HLA-DRA,HLA-DRB1*0401)39-2Kito
Name: transgene insertion 39-2, Kouichi Ito
MGI accession ID: MGI:4429540
Generation method: Transgenic
Attributes: Inserted expressed sequence; Humanized sequence
Synonyms: DR4; DR4-H2E; DRA/DRB1*0401-IE Tg; HLA-DR4; HLA-DR4-IE; HLA-DRA-IEalpha/HLA-DRBI*0401-IEbeta Tg; Tg(HLA-DRA,HLA-DRB1)39-2Kito; Tg(HLA-DRA/H2-Ea,HLA-DRB1*0401/H2-Eb)1Kito
Marker symbol: Tg(HLA-DRA,HLA-DRB1*0401)39-2Kito
Marker name: transgene insertion 39-2, Kouichi Ito
Marker synonyms: Tg(HLA-DRA,HLA-DRB1)39-2Kito
Chromosome: UN
Strain of origin: C57BL/6
Expressed genes: HLA-DRA,major histocompatibility complex, class II, DR alpha,human; HLA-DRB1,major histocompatibility complex, class II, DR beta 1,human
Molecular note: A chimeric MHC class II complex was created by fusing the human peptide antigen binding domains of class II alpha and beta chains (HLA-DRA and HLA-DRBI*0401) to the remaining domains of murine class II alpha or beta chains (H2-Ea and H2-Eb1 genes that make up mouse I-Ed). The chimeric gene constructs were manipulated by replacing exon 2 of IE^d-alpha or IE^d-beta genomic DNA with gene fragments containing exon 2 of HLA-DRA or HLA-DRB1*0401 respectively. The native murine promoters were left intact. These transgene constructs were co-injected into blastocytes. Flow cytometry analysis indicates that B220+ cells co-express the chimeric MHC class II molecules. Line 39-2 is a representative of several lines generated. The chosen human alleles represent human serotype HLA-DR4.

Allele

Symbol: Rag1^tm1Mom
Name: targeted mutation 1, Peter Mombaerts
MGI accession ID: MGI:1857241
Generation method: Targeted
Attributes: Null/Knockout
Synonyms: Rag-; Rag^1tm1Mom; RAG-1-; Rag1^-; RAG1^null; Rag-1KO
Marker symbol: Rag1
Marker name: recombination activating gene 1
Marker synonyms: RAG-1; RNF74
Chromosome: 2
Strain of origin: 129S7/SvEvBrd-Hprt1⁺
Site of expression: expression is seen in bone marrow derived cell lines.
Molecular note: A 1356 bp genomic fragment of the Rag1 gene, encoding the nuclear localization signal and the zinc-finger motif, was replaced by a neomycin cassette. A mutant transcript expressed from this allele was detected by Northern blot in bone marrow derived cell lines from homozygous mice.

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.