C57BL/6J-Ace2^em1Pbmj/J

Stock number: 034903
Product name: Ace2^H353K
Research areas: Developmental Biology Research, Immunology, Inflammation and Autoimmunity Research, Internal/Organ Research, Research Tools, Virology Research

Description

Ace2^H353K knock-in mice have the histidine to lysine amino acid substitution in the angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 locus. In humans, ACE2 is the receptor used for cellular entry by several coronaviruses, including severe acute respiratory syndrome coronavirus-1 (SARS-CoV) and -2 (SARS-CoV-2). These Ace2^H353K knock-in mice show variable susceptibility to to SARS-CoV-2 variants (see Detailed Description), and may be useful for studying antiviral therapies to COVID-19.

Detailed description

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.

Ace2^H353K knock-in mice were created using CRISPR/Cas9 genome editing to express the H353K (histidine to lysine) amino acid substitution in the mouse 353 codon of the Ace2 (angiotensin I converting enzyme (peptidyl-dipeptidase A) 2) locus on the X chromosome. The H353 mutation has been found to confer susceptibility to SARS-CoV-2 infection in vivo.

Ace2 encodes a dipeptidyl carboxydipeptidase involved in cleavage of angiotensin I and II. Mouse Ace2 has high homology to human ACE2. Human ACE2 is the receptor used for cellular entry by several coronaviruses, including severe acute respiratory syndrome coronavirus-1 (SARS-CoV), severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2; 2019 novel coronavirus) and HCoV-NL63. A lysine is present at position 353 of ACE2 in humans, NHP, bats, and ferrets while mice and rats have a histidine at this position.

Prior to infection, Ace2^H353K heterozygous females and hemizygous males are viable and fertile with no obvious phenotype. The viability/fertility/infectivity of homozygous females has not yet been assessed [May 2021].

The donating investigator, Dr. Paul McCray, reports Ace2^H353K mice show that the introduction of a lysine residue at this position in mouse Ace2 results in increased SARS-CoV-2 pseudovirus transduction in vitro and confers susceptibility to SARS-CoV-2 (USA-WA1 isolate) infection and replication in vivo, however Ace2^H353K mice do not develop disease from human virus infection. Disease was achieved in Ace2^H353K mice via infection with SARS-CoV-2 passage 30 variants as described below.

As of May 2021, Dr. McCray reports a SARS-CoV-2 strain that has been serially passaged through Ace2^H353K heterozygotes 30 times (P30). The resulting P30 mouse-adapted virus acquired 6 new mutations, including 2 in the spike protein (K417E, Q493K) predicted to increase the stability and affinity with the receptor. In Ace2^H353K mice (heterozygous females and hemizygous males), intranasal infection with P30 resulted in virulent and lethal disease in older Ace2^H353K mice (6-8 months), whereas younger Ace2^H353K mice exhibit only mild weight loss.
Furthermore, a plaque purified P30 mouse-adapted virus (strain 6-1-1) was then identified. In Ace2^H353K mice (heterozygous females and hemizygous males), intranasal infection with strain 6-1-1 (1x10^3 - 1x10^5 PFU) caused dose-dependent virulence with morbidity/lethality. Specifically, 6-8 month old Ace2^H353K mice showed complete lethality by 6 days post infection (p.i.) at all strain 6-1-1 inocula tested, with males more susceptible than females. The 4 month old Ace2^H353K mice showed nearly complete lethality by 6 days p.i. at all strain 6-1-1 inocula tested.
Of note, wildtype C57BL/6 mice are susceptible to strain 6-1-1 intranasal infection, exhibiting weight loss and dose-dependent lethality by 6 days p.i. Specifically for 7 month old wildtype C57BL/6 mice, 1x10^5 PFU caused complete lethality in males and ~40% lethality in females, 1x10^4 PFU caused ~60% lethality in males (but not females) and 1x10^3 PFU caused ~40% lethality in males (but not females). The 4 month old wildtype C57BL/6 mice exhibited initial weight loss but no lethality.

Researchers may contact Dr. Paul McCray to request the plaque purified P30 mouse-adapted SARS-CoV-2 (strain 6-1-1).

Development

CRISPR/Cas9 endonuclease-mediated genome editing was used to create an amino acid substitution at position 353 (histidine to lysine, H353K, CAC to AAA) in exon 8 (isoform Ace2-201) of the Ace2 locus on the X chromosome. Single guide RNA and Cas9 endonuclease were introduced into C57BL/6J-derived fertilized eggs by pronuclear injection. Embryos were transferred to pseudopregnant females, and correctly targeted pups were bred to C57BL/6J mice for germline transmission. The mutation was confirmed by sequencing. The colony was maintained on the C57BL/6J background. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes.

Allele

Symbol: Ace2^em1Pbmj
Name: endonuclease-mediated mutation 1, Paul McCray
MGI accession ID: MGI:6491923
Generation method: Endonuclease-mediated
Attributes: Not Applicable
Synonyms: Ace2^H353K
Marker symbol: Ace2
Marker name: angiotensin I converting enzyme (peptidyl-dipeptidase A) 2
Marker synonyms: 2010305L05Rik; ACEH
Chromosome: X
Strain of origin: C57BL/6J
Molecular note: CRISPR/cas9 endonuclease-mediated genome editing is used to create an amino acid substitution at position 353 (histidine to lysine, H353K, CAC to AAA) in exon 8 (isoform Ace2-201). The mutation was confirmed by sequencing.