B6.Cg-Tg(K18-ACE2)2Prlmn/J

Stock number: 034860
Product name: K18-hACE2
Research areas: Developmental Biology Research, Immunology, Inflammation and Autoimmunity Research, Internal/Organ Research, Mouse/Human Gene Homologs, Research Tools, Virology Research

Description

K18-hACE2 transgenic 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 are susceptible to SARS-CoV-2 and SARS-CoV viruses, they are useful for studying antiviral therapies to COVID-19 and SARS.

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.

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Detailed description

The Jackson Laboratory has been informed by multiple independent investigators testing these K18-hACE2 mice that, similar to SARS-CoV infection, intranasal infection with SARS-CoV-2 virus results in K18-hACE2 mice with severe illness - reaching criteria for euthanasia ~5-8 days post-challenge [Zheng et al. 2021 Nature 589:603; 1x10^5 PFU] [June 2020 data from Drs. Perlman and McCray]. This was evidenced by body weight loss, rapid breathing, hunched posture and inactivity as result of infection. Virus titers were reportedly detected in the lungs, brain, kidney, liver and spleen (see additional organs below). Gross pathology of the lungs indicates lesions as a result of infection. Vehicle or untreated control mice showed no signs of illness - gaining weight with a normal appearance.
In addition, K18-hACE2 mice infected with SARS-CoV-2 may replicate the chemokine/cytokine storm traits observed in humans [Oladunni et al. 2020 Nat Commun 11:6122; 1x10^5 PFU], and may exhibit viral replication in gut [Rathnasinghe et al. 2020 Emerg Microbes Infect; 1x10^4 PFU] and heart [Winkler et al. 2020 Nat Immunol 21:1327; 2.5x10^4 PFU].
Importantly, some K18-hACE2 animals given lower dose SARS-CoV-2 (2x10^3 PFU) survived despite significant weight loss [Golden et al. 2020 JCI Insight 5:e142032].

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.

Homozygous mice are viable and fertile.

These K18-hACE2 mice develop a rapidly lethal infection after intranasal inoculation with a human strain of SARS-CoV. Infection begins in airway epithelia, with subsequent alveolar involvement and extrapulmonary virus spread to the brain. Infection results in macrophage and lymphocyte infiltration in the lungs and upregulation of proinflammatory cytokines and chemokines in both the lung and the brain. By days 3 to 5 postinfection, K18-hACE2 mice begin to lose weight and become lethargic with labored breathing. Mice from this founder line are moribund 4 days after inoculation, and all mice are dead 7 days after inoculation. Transgenic expression of hACE2 in epithelia converts a mild SARS-CoV infection into a rapidly fatal disease. [McCray et al. 2007 J Virol. 81:813 (PMID:17079315)]

Please note, these K18-hACE2 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.

As with any mutant/transgenic mouse line, researchers are strongly encouraged to genotype the mice they receive to confirm expected zygosity and to confirm the genotyping assay works in their laboratory.

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, 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 this congenic colony at The Jackson Laboratory.

In May 2020, analysis commissioned by The Jackson Laboratory 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).

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: 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.