B6.Cg-Tg(FCGRT)32Dcr Fcgrt^tm1Dcr Ighg1^{em2(IGHG1)Mvw}/MvwJ

Stock number: 029686
Product name: Tg32-hFc , HuPK™
Strain synonyms: Tg32-hFc
Research areas: Immunology, Inflammation and Autoimmunity Research, Research Tools

Description

Tg32-hFc mice carry a humanized FCGRT gene (null for mouse Fcgrt) and a knock-in/knock-out of the Ighg1 locus that replaces the mouse hinge and Fc domain with the equivalent human IgG1 domains. Mice produce human IgG1 Fc-mouse IgG Fab₂ chimeric antibodies at physiological levels and provide a platform for the assessment of human IgG-based biologics.

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Detailed description

Tg32-hFc mice harbor a knock-out (KO) allele of the Fc receptor, IgG, alpha chain transporter (Fcgrt^tm1Dcr), express a human FcRn α-chain (FCGRT) transgene under control of the human FcRn promoter, and contain a CRISPR/Cas9-mediated knock-in/knock-out of the Ighg1 (immunoglobulin heavy constant gamma 1 (G1m marker)) locus that replaces the mouse hinge and Fc domain with the equivalent human IgG1 domains (Ighg1^{em2(IGHG1)Mvw}).

Mice homozygous for all three alleles do not exhibit any gross abnormal phenotype and are viable and fertile. Tg32-hFc mice produce human IgG1 Fc-mouse IgG Fab₂ chimeric antibodies in serum concentrations at physiologically relevant levels, as such they provide an endogenous source of competing human IgG1. Immunization with DNP-KLH (2,4-dinitrophenyl-keyhole limpet hemocyanin) significantly increases the chimeric IgG1 levels. This IgG1 FCGRT humanized mouse model provides a platform for assessment of human IgG-based biologics.

Development

CRISPR/Cas9 endonuclease-mediated genome editing was used to replace the mouse Ighg1 locus hinge (exon 2), CH2 (exon 3) and CH3 (exon 4) regions with a 902 bp human hinge (exon 2), CH2 (exon 3) and CH3 (exon 4) sequences while preserving the splice junctions. Single guide RNAs and cas9 endonuclease were introduced into B6.Cg-Fcgrt^tm1Dcr Tg(FCGRT)32Dcr/DcrJ-derived (Stock No. 014565, abbreviated as Tg32) fertilized eggs by pronuclear injection. Correctly targeted pups were bred to Tg32 mice for germline transmission and the gene modification confirmed by RT-PCR and Sanger sequencing. Mice were interbred to generate homozygotes for all three alleles. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize Tg32 oocytes.

Stock No. 014565 FcRn-/- hFcRn (32) Tg mice have a knock-out mutation for the mouse Fc receptor, IgG, alpha chain transporter gene (Fcgrt) on chromosome 7 and a transgene expressing the human FCGRT gene under the control of its own native promoter (hTg32) on chromosome 2 (101,081,712-101,081,713; mouse genome mm10). See Stock No. 014565 for more information on transgene copy number and other human genes in this transgene.

Allele

Symbol: Ighg1^{em2(IGHG1)Mvw}
Name: endonuclease-mediated mutation 2, Michael V Wiles
MGI accession ID: MGI:6467517
Generation method: Endonuclease-mediated
Attributes: Inserted expressed sequence; Humanized sequence
Synonyms: hFC
Marker symbol: Ighg1
Marker name: immunoglobulin heavy constant gamma 1 (G1m marker)
Marker synonyms: IgG1; IgG3; Igh-4
Chromosome: 12
Strain of origin: B6.Cg-Fcgrt^tm1Dcr Tg(FCGRT)32Dcr/DcrJ
Molecular note: CRISPR/cas9 endonuclease-mediated genome editing is used to replace the mouse Ighg1 locus hinge (exon 2), CH2 (exon 3) and CH3 (exon 4) regions with a 902 bp human hinge (exon 2), CH2 (exon 3) and CH3 (exon 4) sequences while preserving the splice junctions.

Allele

Symbol: Fcgrt^tm1Dcr
Name: targeted mutation 1, Derry C Roopenian
MGI accession ID: MGI:3511510
Generation method: Targeted
Attributes: Null/Knockout
Synonyms: FcRn^-
Marker symbol: Fcgrt
Marker name: Fc receptor, IgG, alpha chain transporter
Marker synonyms: alpha-chain; FcgammaRn; FCRN; neonatal Fc receptor
Chromosome: 7
Strain of origin: 129X1/SvJ
Molecular note: Sequence from exon 1 and part of exon2 was replaced with a PGK-neo cassette. Quantitative PCR of liver cDNA indicated the absence of mRNA. Western blot analysis of neonatal intestinal extracts failed to reveal protein product.

Allele

Symbol: Tg(FCGRT)32Dcr
Name: transgene insertion 32, Derry C Roopenian
MGI accession ID: MGI:4944199
Generation method: Transgenic
Attributes: Inserted expressed sequence; Humanized sequence
Synonyms: hFcRn(32) vhFcRn (32) Tg hFcRn (32) Tg; Tg32
Marker symbol: Tg(FCGRT)32Dcr
Marker name: transgene insertion 32, Derry C Roopenian
Chromosome: 2
Strain of origin: C57BL/6J
Expressed genes: FCGRT,Fc fragment of IgG receptor and transporter,human
Site of expression: The transgene expresses human FCGRT from the native human promoter, and displays a physiological human FCGRT expression pattern (Latvala et al., 2017).
Molecular note: A 34Kb fragment of a BAC containing the entire human FCGRT , Fc fragment of IgG, receptor, transporter, alpha, gene (approximately 11kb) and approximately 10kb of flanking sequence was sublconed into a SuperCos vector. The resulting cosmid was microinjected into fertilized C57BL/6J mouse eggs. Founder line 32 was established. Transgene insertion occurred on Chr 2.