C57BL/6J-Gt(ROSA)26Sor^{em25(KRT18-ACE2)Mvw}/MvwJ

Stock number: 036363
Product name: B6.RMCE[K18-HuACE2]
Strain synonyms: B6.RMCE[Krt18-ACE2], B6.RMCE[K18-HuACE2]
Research areas: Immunology, Inflammation and Autoimmunity Research, Mouse/Human Gene Homologs, Research Tools, Virology Research

Description

B6.RMCE[K18-HuACE2] 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 epithelial cells, including airway epithelia where infections typically begin. Because K18-HuACE2 mice are susceptible to SARS-CoV-2 and SARS-CoV viruses, they may be useful for studying antiviral therapies to COVID-19 and SARS.

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.

B6.RMCE[K18-HuACE2] mice express human ACE2, the receptor used by severe acute respiratory syndrome coronavirus (SARS-CoV) to gain cellular entry and a C-terminal FLAG epitope tag. The human keratin 18 promoter directs expression to epithelial cells, including airway epithelia where infections typically begin. Because K18-HuACE2 mice are susceptible to SARS-CoV-2 and SARS-CoV viruses, they may be useful for studying antiviral therapies to COVID-19 and SARS.

The B6.RMCE[K18-HuACE2] mice were made using Bxb1 integrase (Bxb1 Int)-mediated transgenesis and the RCME-ready zygotes carrying the RosaBxb-GT/GA "safe harbor landing pad" in the first intron of Gt(ROSA)26Sor. This method excludes the donor backbone and precisely integrates the donor sequence into the Gt(ROSA)26Sor locus (Low et al., 2022). This RMCE[K18-HuACE2] allele (attR-GT::KRT18-ACE2::attL-GA) is designed such that further exposure to Bxb1 integrase alone should not delete/invert/alter the KRT18-ACE2 sequence. Mice homozygous for the mutation are viable and fertile.

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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 B6.RMCE[K18-HuACE2] allele was generated in the laboratory of Dr. Michael V. Wiles (The Jackson Laboratory). RMCE (recombinase-mediated cassette exchange) was utilized to insert a 6.84 kb constuct containing a human keratin 18 promoter directing expression of full length human ACE2 followed a C-terminal FLAG epitope tag into a modified Gt(ROSA)26Sor (B6.RosaBxb-GT/GA) locus on chromosome 6. The C57BL/6J-derived zygotes carrying the Gt(ROSA)26Sor^em7Mvw allele (RosaBxb-GT/GA, from Stock No. 036152) were previously targeted to insert a dual Bxb1 attP-site "safe harbor landing pad" in the first intron of Gt(ROSA)26Sor. The landing pad contains a 48 bp Bxb1 attP-GT sequence and a 48 bp Bxb1 attP-GA sequence separated by a 240 bp spacer of native Gt(ROSA)26Sor sequence. The DNA donor plasmid, carrying two heterologous attB sites and donor transgene, was then microinjected with Bxb1 integrase into the RMCE-ready zygotes (attP-GT/GA). The resulting offspring with the correctly targeted Gt(ROSA)26Sor allele RMCE[K18-HuACE2] (attR-GT::KRT18-ACE2::attL-GA) were then backcrossed to C57BL/6J. The transgene was confirmed by PCR and Sanger sequencing and verified by nCATS (nanopore Cas9-targeted sequencing). The colony was maintained on the C57BL/6J background. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

Allele

Symbol: Gt(ROSA)26Sor^{em25(KRT18-ACE2)Mvw}
Name: endonuclease-mediated mutation 25, Michael V Wiles
MGI accession ID: MGI:6877060
Generation method: Endonuclease-mediated
Attributes: Inserted expressed sequence; Humanized sequence; Epitope tag
Synonyms: RMCE[K18-HuACE2]
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Marker synonyms: beta geo; Gtrgeo26; Gtrosa26; R26; ROSA26; SETD5-AS1; Thumpd3as1
Chromosome: 6
Strain of origin: C57BL/6J
Molecular note: CRISPR/cas9 genome editing and RMCE (recombinase-mediated cassette exchange) are utilized to insert a human keratin 18 promoter directing expression of human ACE2 followed a C-terminal FLAG epitope tag into a modified Gt(ROSA)26Sor (B6.RosaBxb-GT/GA) locus. The C57BL/6J-derived zygotes carrying the Gt(ROSA)26Sor (RosaBxb-GT/GA) allele were previously targeted to insert a dual Bxb1 attP-site "safe harbor landing pad" in the first intron of Gt(ROSA)26Sor. The landing pad contains a 48 bp Bxb1 attP-GT sequence and a 48 bp Bxb1 attP-GA sequence separated by a 240 bp spacer of native Gt(ROSA)26Sor sequence. The DNA donor plasmid, carrying two heterologous attB sites and donor transgene, was then microinjected with Bxb1 integrase into the RMCE-ready zygotes (attP-GT/GA).