NOD.Cg-Rag1^tm1Mom Flt3^tm1Irl Mcph1^{Tg(HLA-A2.1)1Enge} Il2rg^tm1Wjl/J

Stock number: 033127
Product name: NFA2, NFA2-HIS, NRG-Flk2-/- HLA-A*0201

Research areas: Cancer Research, Hematological Research, Immunology, Inflammation and Autoimmunity Research, Internal/Organ Research, Research Tools

Description

NFA2 mutant mice expand the capabilities of NRG (NOD.Cg-Rag1^tm1Mom Il2rg^tm1Wjl) strain by incorporating the HLA-A2.1 human major histocompatibility transgene and an Flt3 (FMS-like tyrosine kinase 3; also called Flk2) knock-out allele that impairs the development of native mouse myeloid cells.

Detailed description

NRG (NOD.Cg-Rag1^tm1MomIl2rg^tm1Wjl; see stock# 007799) is a commonly-used humanized mouse strain that is useful in studying a broad range of human-tropic pathogens by virtue of their inability to develop functional murine B, T, or natural killer (NK) cells. The NOD genetic background contributes C5 complement deficiency in addition to a Sirpa (signal-regulatory protein alpha) polymorphism which reduces phagocytic activity against human cells. Injection of irradiation-conditioned NRG mice with human hematopoietic stem cells (HSCs) leads to de novo hematopoiesis, resulting in stable engraftment of human hematolymphoid system components.

These NFA2 mutant mice expand the capabilities of the NRG strain by incorporating an HLA-A2.1 human major histocompatibility transgene (Mcph1^{Tg(HLA-A2.1)1Enge}; see stocks 003475 and  005512) and an Flt3 (FMS-like tyrosine kinase 3; also called Flk2) targeted knock-out allele. The Flt3 mutation impairs the development of native mouse myeloid cells which can potentially interfere with human myelopoiesis, as well as with the priming of human-specific adaptive immune responses upon infection with human-tropic pathogens.

As compared to humanized NRG mice, humanized NFA2 mice treated with FMS-like tyrosine kinase 3 ligand (Flt3LG) demonstrate an upregulation of a broad range of human-specific myeloid and NK cell functional and activation markers in response to yellow fever virus vaccine (YFV-17).  These markers include: HLA-DRA, CD83, and CD40 for cDCs, and CCL5, GNLY, GZMA, PRF1, and CD226 for NK cells. Increased myeloid reconstitution and activation in these mice correlated with enhanced T-cell specific response and control of viral infection. Additionally, the human transcriptomic signature associated with YFV-17D vaccination in the peripheral blood of humanized NFA2 mice displayed superior overlap with the blood transcriptomic signature observed in human vaccinees in comparison to conventional humanized NRG mice.

Created as part of a study measuring the quality of human immune responses in humanized mouse models, these mice enable further studies of human-specific adaptive responses within the context of a functional myeloid compartment in humanized mice.

Development

The Flt3^tm1Irl (Flk2^-) mutation was generated by the insertion of a neomycin cassette in exon 3 of the Flt3 gene via homologous recombination in 129S/SvEv-Gpi1^c-derived CCE/EK.CCE embryonic stem (ES) cells. RNAse protection assays detect low levels of transcript in homozygous mutant mice. RT-PCR and sequence analyses of the mutant transcript show that the targeted exon has been excised, resulting in a frameshift mutation that introduces a stop codon after codon 51 and renders the protein non-functional.

Flt3^tm1Irl mice were bred with NRG mice (NOD.Cg-Rag1^tm1MomIl2rg^tm1Wjl/SzJ; see Stock No. 007799) for ten generations by the donating laboratory to produce NRGF animals (NRG-Flk2-/-).

NOD.Cg-Prkdc^scid Il2rg^tmlWjl Mcph1^{Tg(HLA-A2.1)1Enge} mice (NSG-A2*0201) were also crossed with NRG mice to create NOD background NRG-A2 animals carrying the Il2rg knock-out allele and the HLA-A2.1 transgene. In brief, the Mcph1^{Tg(HLA-A2.1)1Enge} mutation (see Stock Nos. 003475 and 005512) involves the introduction of full length HLA-A2.1 to mouse Chromosome 8, which caused a duplication of the Mcph1 (microcephaly, primary autosomal recessive 1) gene. Prkdc^scid was bred out of the NRG-A2 line.

The NRGF and NRG-A2 animals were then intercrossed by the donating laboratory to produce NFA2 mice (NFA2-HIS; NRG-Flk2-/- HLA-A*0201) carrying four mutations on the NOD genetic background: NOD-Rag1^tm1Mom Flt3^tm1Irl Mcph1^{Tg(HLA-A2.1)1Enge} Il2rg^tm1Wjl.

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: Flt3^tm1Irl
Name: targeted mutation 1, Ihor R Lemischka
MGI accession ID: MGI:2384051
Generation method: Targeted
Attributes: Null/Knockout
Synonyms: Flk2^-
Marker symbol: Flt3
Marker name: FMS-like tyrosine kinase 3
Marker synonyms: CD135; FLK2; FLK-2; Flt-3; STK1; wmfl
Chromosome: 5
Strain of origin: 129S/SvEv-Gpi1^c
Molecular note: Exon 3 was disrupted by the insertion of a neomycin selection cassette at the codon corresponding to residue 59 in the mature protein. An RNAse protection assay detected low levels of transcript in homozygous mutant mice. RT-PCR and sequence analyses of the mutant transcript showed that the targeted exon 3 had been excised, resulting in a frameshift mutation that introduces a stop codon after codon 51.

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: Mcph1^{Tg(HLA-A2.1)1Enge}
Name: transgene insertion 1, Victor H Engelhard
MGI accession ID: MGI:3056375
Generation method: Transgenic
Attributes: Null/Knockout; Inserted expressed sequence; Humanized sequence
Marker symbol: Mcph1
Marker name: microcephaly, primary autosomal recessive 1
Marker synonyms: 5430437K10Rik; BRIT1; D030046N04Rik; MCT; RGD1560642
Chromosome: 8
Strain of origin: C57BL/6
Expressed genes: HLA-A,major histocompatibility complex, class I, A,human
Molecular note: The transgene contains the full length HLA-A2.1 gene. Expression of human class I MHC Ag HLA-A2.1 was detected on cells from homozygous transgenic spleen, bone marrow, and thymus. Line 1 inserted into the gene at 18736683-18757058 (Build GRCm38/mm10) resulting in a duplication. The duplication results in a functional knock-out. Founder line 1 has a copy number of 3-8.
General note: Expression of this human class I molecule in transgenic mice does not result in expansion of the number of cytotoxic T lymphocyte (CTL) precursors specific for other human class I Ag, HLA-B27 or HLA-A2.2.