B6.Cg-Fcgrt^tm1Dcr Tg(FCGRT)32Dcr/DcrJ

Stock number: 014565
Product name: FcRn-/- hFcRn (32) Tg , hFcRn Tg32 , Tg32 , HuPK™
Strain synonyms: FcRn^-/- hFcRn (32) T
Research areas: Immunology, Inflammation and Autoimmunity Research, Research Tools, Virology Research

Description

Tg32 mice (also called hFcRn Tg32 or FcRn-/- hFcRn line 32 Tg) have the mouse Fcgrt knock-out mutation and a transgene expressing the human FCGRT gene under the control of its own native promoter (hTg32). Useful in evaluating the pharmacokinetics and pharmacodynamics of human immunoglobulin G (IgG) and Fc-domain based therapeutics, homozygous Tg32 mice have the highest, most human-like protection of humanized IgG and are the best model for use when maximum half-life data is required.

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In Goodwin et al. 2019 Genome Res. 29:494, analysis performed on one mouse from Stock No. 014565 estimated that Tg(FCGRT)32Dcr has more than 20 transgene copies integrated on chromosome 2 [101,081,712-101,081,713; mouse genome mm10]. Within the transgene, the human sequence Chr19:50,000,289-50,033,653 [human genome hg19] was identified, which contains the entire FCGRT, RPS11, SNORD35B and MIR150 human genes, and part of the RCN3 human gene. No deletion of mouse genome was identified near the insertion site.

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.

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).

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 vivoTg276 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.

Development

These knock-out/transgenic mice were generated in the laboratory of Dr. Derry Roopenian (The Jackson Laboratory) by crossing Fcgrt^tm1Dcr mutant mice with Tg(FCGRT)32Dcr transgenic mice (coisogenic on a C57BL/6J genetic background). These double mutant mice were backcrossed for 11 generations to C57BL/6J prior to transfer to The Jackson Laboratory Repository.

To generate the FcRn α-chain knock-out 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) of the Fc receptor, IgG, alpha chain transporter gene (Fcgrt) on chromosome 7 with a PGK-Neo^r cassette. The vector was electroporated into 129/SvJ-derived ESV/J-1182 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts. The resulting chimeric animals were crossed to C57BL/6J mice.

To generate the hFcRn line 32 transgenic mice, a 33-Kb cosmid clone including the complete human FCGRT gene (approximately 11kb as well as 10kb of 5' and 3' flanking sequences) was microinjected into fertilized C57BL/6J mouse eggs. Founder line 32 was established. Transgene insertion occurred on mouse Chromosome 2.

In Goodwin et al. 2019 Genome Res. 29:494, analysis performed on one mouse from Stock No. 014565 estimated that Tg(FCGRT)32Dcr has more than 20 transgene copies integrated on chromosome 2 [101,081,712-101,081,713; mouse genome mm10]. Within the transgene, the human sequence Chr19:50,000,289-50,033,653 [human genome hg19] was identified, which contains the entire FCGRT, RPS11, SNORD35B and MIR150 human genes, and part of the RCN3 human gene. No deletion of mouse genome was identified near the insertion site.

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.