B6.Cg-Tg(K18-ACE2)2Prlmn Tg(FCGRT)32Dcr Fcgrt^tm1Dcr/J

Stock number: 034902
Product name: K18-hACE2 ; FcRn-/- hFcRn (32) Tg
Research areas: Developmental Biology Research, Immunology, Inflammation and Autoimmunity Research, Internal/Organ Research, Research Tools, Virology Research

Description

These K18-hACE2 ; FcRn-/- hFcRn (32) Tg mice have the K18-hACE2 transgene and hFcRn (32) transgene in trans (on separate chromosome 2 homologs), whereas Stock No. 037043 has the two transgenes in cis (on the same chromosome 2 homolog).

These mice express human ACE2, the receptor used by severe acute respiratory syndrome coronavirus (SARS-CoV) to gain cellular entry. The human keratin 18 promoter directs expression to epithelia, including airway epithelia where infections typically begin. Because K18-hACE2 mice are susceptible to SARS-CoV-2 and SARS-CoV viruses, they are useful for studying antiviral therapies to COVID-19 and SARS.

This strain also has the FcRn-/- targeted mutation and hFcRn (32) transgene, which may enhance the human-like protection of humanized IgG and potentially make these mice useful for developing antibody-based therapeutics against SARS-CoV, including assessing half-life, selecting efficacious variants and determining dosage for clinical trials.

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Detailed description

Please see the K18-hACE2 datasheet (Stock No. 034860) for important safety/handling and any updated information on K18-hACE2 mice in SARS-CoV-2 research.
Please note, these mice should be handled in a manner consistent with CDC/ABSA/WHO guidelines for prevention of human infection with the SARS-CoV-2 virus. Proper PPE and handling methods should be used at all times when working with these mice. Additional important guidelines for using SARS-CoV-2 susceptible mouse lines.

Stock No. 034902 K18-hACE2 ; FcRn-/- hFcRn (32) Tg is a multiple mutant strain assembled by breeding Stock Nos. 034860 and 014565. Stock No. 034902 has the K18-hACE2 transgene and hFcRn (32) transgene in trans (on separate chromosome 2 homologs). In an attempt to offer popular alleles together, mouse lines may be bred together for purpose of researchers to study and characterize. It should be noted that the phenotype of Stock No. 034902 could vary from that originally described for the individual mouse lines used to assemble it. We may modify the strain description if necessary as published results become available. Each mutant allele is briefly described below [September 2020].

K18-hACE2 transgenic mice express the receptor for severe acute respiratory syndrome (SARS), caused by a coronavirus (SARS-CoV) in airway and other epithelia under control of the human keratin 18 (KRT18) promoter. Specifically, these mice contain 2.5 kb of the KRT18 genomic sequence, including the promoter, and the first intron (with a mutation in the 3' splice acceptor site to reduce exon skipping) and a translational enhancer (TE) sequence from alfalfa mosaic virus, upstream of the human angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 coding sequence (hACE2), followed by exons 6-7 and the poly(A) signal of the human K18 gene. These elements have been found to be necessary for high-level expression and epithelial cell specificity of hACE2, the primary host cell receptor for SARS-CoV. In these mice, from founder line 2, K18-hACE2 transgene expression is evident in airway epithelial cells (but not in alveolar epithelia), as well as in epithelia of other internal organs, including the liver, kidney, and gastrointestinal tract. Recent research indicates that this line may also be useful in studies related to the study of 2019 novel coronavirus (SARS-CoV-2) pathogenesis.

Addition of the FcRn-/- targeted mutation and hFcRn (32) transgene may enhance the human-like protection of humanized IgG potentially making these mice a good model for developing antibody-based therapeutics against SARS-CoV, including assessing half-life, selecting efficacious variants, and determining dosage for clinical trials. Using this model, clinicians may be able to estimate the minimum antibody dose to achieve therapeutic serum concentrations, reducing the need for potentially risky dose-escalation treatments during clinical trials.

The K18-hACE2 transgenic line and the FcRn-/- targeted mutation and hFcRn (32) transgenic line are able to be maintained as homozygous in their individual mouse lines. However, because the two transgenes are located ~2 Mbp apart on separate chromosome 2 homologs (in trans), it may be difficult to generate mice with the two transgenes in cis (on the same chromosome 2 homolog). As such, The Jackson Laboratory will maintain and distribute Stock No. 034902 as hemizygous for the K18-hACE2 transgene, hemizygous for the hFcRn (32) transgene and homozygous for the FcRn-/- targeted mutation.

Development

The ~6.8 kb K18-hACE2 transgene was designed with, from 5' to 3', 2.5 kb of genomic sequence, including the promoter, and the first intron (with a mutation in the 3' splice acceptor site to reduce exon skipping) from the human keratin 18 (KRT18) gene, and a translational enhancer (TE) sequence from alfalfa mosaic virus. This was followed by the human angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 (hACE2) coding sequence, and exons 6-7 and the poly(A) signal of the human K18 gene. This transgene was microinjected into fertilized (C57BL/6J x SJL/J)F2 oocytes. K18-hACE2 mice from founder line 2, with 8 copies of the transgene (see additional detail below), were subsequently maintained by breeding transgenic mice with C57BL/6 mice for at least two generations (the genetic background of the mice sent to The Jackson Laboratory was later determined to be C57BL/6N via SNP testing). Upon arrival, three backcrosses to C57BL/6J (Stock No. 000664) were then performed to rederive, establish and expand a congenic colony at The Jackson Laboratory as Stock No. 034860.

To create Stock No. 034902, some mice from Stock No. 034860 were bred to FcRn-/- hFcRn (32) Tg mice (Stock No. 014565) carrying 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 bp).
Because the two transgenes are located ~2 Mbp apart on separate chromosome 2 homologs (in trans), it may be difficult to generate Stock No. 034902 mice with the two transgenes in cis (on the same chromosome 2 homolog). As such, The Jackson Laboratory will maintain and distribute Stock No. 034902 as hemizygous for the K18-hACE2 transgene, hemizygous for the hFcRn (32) transgene and homozygous for the FcRn-/- targeted mutation.

In May 2020, analysis commissioned by The Jackson Laboratory on Stock No. 034860 determined that multiple copies of the Tg(K18-ACE2)2Prlmn transgene integrated at a single site on chromosome 2 (99,209,508-99,220,724; mouse mm10). An ~11 kb duplication of the host genome (chr2:99,209,508-99,220,724) is present at both ends of the integrated transgene array. Currently, there are no genes annotated in this region. The transgene copy number of the hemizygous mouse was estimated to be 8 full copies (or 12-30 partial copies).
Furthermore, in Low et al. 2022 Sci Rep 12:5424 - Figure 6C, nanopore Cas9-targeted sequencing (nCATS) further illustrated the complex architectural nature of the transgenic insertion site on chromosome 2. This identified K18-ACE2 concatemers consisting of at least 11 copies in both orientations, along with K18-ACE2 cassette truncations at sites of inversion throughout the insertion. This also detected an ~10.5 kb duplication of chromosome 2 at both ends of the insertion region.

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.

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
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.

Allele

Symbol: Tg(K18-ACE2)2Prlmn
Name: transgene insertion 2, Stanley Perlman
MGI accession ID: MGI:6389235
Generation method: Transgenic
Attributes: Inserted expressed sequence; Humanized sequence
Marker symbol: Tg(K18-ACE2)2Prlmn
Marker name: transgene insertion 2, Stanley Perlman
Chromosome: 2
Strain of origin: (C57BL/6J x SJL/J)F2
Site of expression: Epithelia, including airway epithelia.
Molecular note: The ~6.8 kb transgene was designed with, from 5' to 3', 2.5 kb of genomic sequence, including the promoter, and the first intron (with a mutation in the 3' splice acceptor site to reduce exon skipping) from the human K18 gene, and a translational enhancer (TE) sequence from alfalfa mosaic virus. This was followed by the human angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 (hACE2) coding sequence (lacking the last exon), and exons 6-7 and the poly(A) signal of the human K18 gene. The transgene integrated at a single site on chromosome 2 (99,209,508-99,220,724; mouse mm10). An ~11 kb duplication of the host genome (chr2:99,209,508-99,220,724) is present at both ends of the integrated transgene array. Currently, there are no genes annotated in this region. The transgene copy number of the hemizygous mouse is estimated to be 8 full copies (or 12-30 partial copies). Transgene expression is evident in airway epithelial cells (but not in alveolar epithelia), as well as in epithelia of other internal organs, including the liver, kidney, and gastrointestinal tract.