C57BL/6-Ace2^em1Brle/J

Stock number: 039339
Product name: mAce2^F83Y,H353K
Research areas: Developmental Biology Research, Immunology, Inflammation and Autoimmunity Research, Internal/Organ Research, Research Tools, Virology Research

Description

mAce2^F83Y,H353K knock-in (KI) mice harbor two mutations, F83Y and H353K, in the endogenous Ace2 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 mAce2^F83Y,H353K KI mice 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 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. mAce2^F83Y,H353K knock-in (KI) mice were created using CRISPR/Cas9 genome editing to express both the F83Y (phenylalanine to tyrosine) and H353K (histidine to lysine) amino acid substitutions in the mouse Ace2 locus on the X chromosome. These mutations have been found to confer susceptibility to SARS-CoV-2 infection in vivo by “humanizing” the ACE2 SARS-CoV-2 spike protein receptor-binding domain (RBD).

Prior to infection, mAce2^F83Y,H353K homozygous females and hemizygous males are viable and fertile with no obvious phenotype. These KI mice demonstrate mAce2^F83Y,H353K expression levels comparable to wildtype Ace2 expression across multiple tissues. When exposed to SARS-CoV-2 via intranasal dosing, these KI mice did not present with clinical symptoms; however, there was a significant viral load in the lungs. These mAce2^F83Y,H353K KI mice may be useful for studying antiviral therapies to COVID-19.

Development

The mACE2^F83Y,H353K knock-in (KI) allele was generated using CRISPR/Cas9 endonuclease-mediated genome editing. Guide RNAs [5'-TTCTTGTAGTGAGAAACTTT (PAM: GGG)-3'(for F83Y) and 5'-ACAGCTTGGGATCTGGGACA (PAM: CGG)-3'(for H353K)] were used to target the angiotensin converting enzyme 2 (Ace2) locus on chromosome X. Ace2 transcript Ace2-202 was used as reference for the exon number and the guide sequences. The sgRNAs, ssDNA and the Cas9 nuclease mRNA were introduced into the cytoplasm of C57BL/6J (Stock No. 000664) zygotes with well recognized pronuclei. Correctly targeted pups were identified by PCR and Sanger sequencing. The donating laboratory backcrossed the mACE2^- KI mice to C57BL/6J for 2 generations to reduce off-target events (see SNP note below). Upon arrival at The Jackson Laboratory, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes.



In 2024, a 48 SNP (single nucleotide polymorphism) panel analysis, with 43 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the males sent to The Jackson Laboratory Repository. All 5 of the markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6N;C57BL/6J genetic background.

Allele

Symbol: Ace2^em1Brle
Name: endonuclease-mediated mutation 1, Brendan Lee
MGI accession ID: MGI:7628004
Generation method: Endonuclease-mediated
Attributes: Humanized sequence
Synonyms: mAce2^F83Y,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: Using CRISPR/Cas9 technology, guide RNAs (sgRNAs) and oligo DNA donors were designed and synthesized to introduce the F83Y and H353K missense substitutions in Ace2.