B6.Cg-Fcgrt^tm1Dcr Tg(CAG-FCGRT)276Dcr/DcrJ

Stock number: 004919
Product name: FcRn KO hFcRn Tg, Tg276

Strain synonyms: FcRn<-/-> hFcRn (276) Tg
Research areas: Immunology, Inflammation and Autoimmunity Research, Research Tools, Virology Research

Description

Tg276 mice (also called FcRn-/- hFcRn (line 276) Tg) carry a knock-out mutation for the mouse Fcgrt (Fc receptor, IgG, alpha chain transporter) gene and a transgene expressing the human FCGRT gene under the control of a widely-expressed CAG promoter. Mice hemizygous for this transgene are best suited to detect subtle differences in antibody persistence in vivo due to less efficient binding between IgGs and FCGRT.

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Detailed description

There are several challenges facing researchers who are developing antibody-based therapeutics, including assessing half-life, selecting efficacious variants, and determining dosage for clinical trials. The mechanism behind IgG's extended half-life lies in the interaction between IgG and the neonatal Fc receptor (FcRn), encoded by Fcgrt, Fc receptor, IgG, alpha chain transporter (Roopenian et al., 2003; Challa et al., 2014). Standard rodent models are problematic in that mouse and rat FcRn do not bind human IgG with the same affinity as human FcRn, leading to inaccurate half-life data (Ober et al., 2001).

Tg32 (Stock No. 014565) and Tg276 (Stock No. 004919) are humanized mouse models that can be used to predict the pharmacokinetics (PK) of IgG antibodies in humans with an accuracy comparable to non-human primates (NHPs). Both carry a knock-out mutation for the mouse Fcgrt gene (Stock No. 003982) in addition to a transgene expressing the human FCGRT gene either under the control of its own native promoter (Stock No. 014565) or under the control of the more broadly-expressed CAG promoter (human cytomegalovirus (CMV) enhancer fused to the chicken beta-actin; Stock No. 004919). Each line has its unique attributes and optimal applications.

Tg276 mice (Stock No. 004919) express the human FCGRT transgene from the broadly-expressed CAG promoter (human cytomegalovirus (CMV) enhancer fused to the chicken beta-actin). Plasma albumin levels are similar to wild-type mice, however the serum IgG levels remain low due to the species-specific behavior of human FCGRT. These mice are best suited to detect subtle differences in antibody persistence in vivo due to less efficient binding between IgGs and FCGRT. While immunoglobulin half-life measurements show an intermediate correlation to human data, the strength of the Tg276 model lies in its ability to allow fine distinctions in half-life between several candidate molecules. Longer IgG half-lives are observed in homozygotes, as compared to hemizygotes. Petkova et al., 2006 demonstrate that antibody half-life in Tg32 hemizygotes (Stock No. 014565) is comparable to that of Tg276 (Stock No. 004919) homozygotes.

Echoviruses, amongst the most common causative agents of aseptic meningitis, do not infect mouse cells efficiently. Morosky, et al., 2019 have demonstrated, however, that expression of human FCGRT (FcRn), as found in Tg276 mice, restores echovirus infection in mouse cells. Expression of human, but not mouse, FCGRT renders non-permissive human and mouse cells sensitive to echovirus infection and the extracellular domain of human FCGRT directly binds echovirus particles and neutralizes infection. Data show that a number of clinically relevant echoviruses commonly associated with human disease, including E9, E30, and E11, utilize FCGRT as a receptor, suggesting a pan-echovirus role. In contrast, FCGRT plays no role in the infection of related enteroviruses, including enterovirus coxsackievirus B3 (CVB) and poliovirus (PV).

Tg32 mice (also called hFcRn Tg32 or FcRn-/- hFcRn line 32 Tg; Stock No. 014565) express the human FCGRT transgene from the native human promoter, and have the highest, most human-like protection of humanized IgG and are the best model for use when maximum half-life data is required. The transgene, integrated on mouse Chromosome 2, displays a physiological human FCGRT expression pattern (Latvala et al., 2017). Experimental variability between the mice is minimal, enabling a small number of animals per study. Using this model and human PK-predictive allometric scaling (Betts et al., 2018), clinicians can estimate the minimum dose to achieve therapeutic serum concentrations, reducing the need for potentially risky dose-escalation treatments during clinical trials. Avery et al., 2016 show that monoclonal antibody clearance in homozygous mice correlates with human pharmacokinetics better than non-human primates. In this line, serum albumin levels are slightly above those seen in C57BL/6J mice, while mouse IgG levels are low due to the species-specific activity of human FCGRT.

Immunodeficient FCGRT-humanized models carrying the Tg32 transgene are also available to evaluate Fc-domain based therapeutics that are potentially immunogenic or involve xenografts - see B6.Cg-Fcgrt^tm1Dcr Prkdc^scid Tg(FCGRT)32Dcr/DcrJ (Stock No. 018441), and NOD.Cg-Fcgrt^tm1Dcr Prkdc^scid Il2rg^tm1Wjl Tg(FCGRT)32Dcr/J (Stock No. 028615).

Development

This knock-out/transgenic strain was generated in the laboratory of Derry Roopenian (The Jackson Laboratory) by crossing Fcgrt^tm1Dcr targeted mutant mice (backcrossed five generations to C57BL/6J) with Tg(CAG-FCGRT)276Dcr transgenic mice (coisogenic on a C57BL/6J genetic background). These double mutant mice were backcrossed for several additional generations to C57BL/6J (~N11) prior to transfer to The Jackson Laboratory Repository.

To generate the FcRn α-chain knockout mice (Fcgrt^tm1Dcr; see also Stock No. 003982), a targeting vector was designed to replace 1588 nucleotide fragments (encoding promoter sequence 5' of the transcriptional start site, exon1, intron 2, and most of exon2) with a PGK-Neo^r cassette. The vector was electroporated into 129X1/SvJ-derived ESV/J-1182 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts. Resultant mice were backcrossed to C57BL/6J for five generations prior to breeding with hFcRn line 276 (Tg276) transgenic mice.

To generate the Tg276 transgenic mutation, a transgenic vector was designed with the 0.34 kb human cytomegalovirus immediate early promoter enhancer, 1.4 kb chicken beta-actin promoter and intron 1, a cDNA sequence encoding the human FcRn α-chain (FCGRT), 0.73 kb rabbit beta-globin intron, and 0.35 kb SV40 polyA sequence. The transgenic construct was injected into C57BL/6J embryos. The resulting transgenic offspring were bred to C57BL/6J mice to establish founder line 276. Mice were maintained on a C57BL/6J genetic background. Transgene insertion occurred on mouse Chromosome 1.

Allele

Symbol: Tg(CAG-FCGRT)276Dcr
Name: transgene insertion 276, Derry C Roopenian
MGI accession ID: MGI:4441040
Generation method: Transgenic
Attributes: Inserted expressed sequence; Humanized sequence
Marker symbol: Tg(CAG-FCGRT)276Dcr
Marker name: transgene insertion 276, Derry C Roopenian
Chromosome: 1
Strain of origin: C57BL/6J
Expressed genes: FCGRT,Fc fragment of IgG receptor and transporter,human
Molecular note: To generate the hFcRn line 276 transgenic mice, a transgene was designed with the 0.34 kb human cytomegalovirus immediate early promoter enhancer, 1.4 kb chicken beta-actin promoter and intron 1, a cDNA sequence encoding the human FcRn alpha-chain, 0.73 kb rabbit beta-globin intron, and 0.35 kb SV40 polyA sequence. Line 276 was established. Transgene insertion occurred on Chr 1, causing a 1 bp duplication.

Allele

Symbol: Fcgrt^tm1Dcr
Name: targeted mutation 1, Derry C Roopenian
MGI accession ID: MGI:3511510
Generation method: Targeted
Attributes: Null/Knockout
Marker symbol: Fcgrt
Marker name: Fc receptor, IgG, alpha chain transporter
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.