C(Cg)-Tmprss2^{em2.1(TMPRSS2)Synbl} Ace2^{em2.1(ACE2)Synbl}/J

Stock number: 037079
Product name: hACE2-hTMPRSS2-BALB/c , ACE2-TMPRSS2-C , ACE2-C;TMPRSS2-C
Research areas: Cancer Research, Developmental Biology Research, Immunology, Inflammation and Autoimmunity Research, Internal/Organ Research, Mouse/Human Gene Homologs, Research Tools, Virology Research

Description

hACE2-hTMPRSS2-BALB/c mice combine the knock-in/knock-out mutations found in BALB/cJ-Ace2^{em2.1(ACE2)Synbl}/J (Stock No. 037077) and BALB/cJ-Tmprss2^{em2.1(TMPRSS2)Synbl}/J (Stock No. 037078). Expression of the mouse Ace2 and Tmprss2 genes is replaced with that of the homologous human genes. This strain has been useful in studies of Covid viral infection.

Detailed description

TMPRSS2 (transmembrane protease, serine 2) is a membrane-associated serine protease, expressed in the epithelium of the human respiratory tract and prostate glands. It has been shown to activate hemagglutinin proteins of human influenza viruses and is implicated in prostate carcinogenesis. SARS-CoV-2 binds to the extracellular receptor angiotensin-converting enzyme 2 (ACE2) to enter the host cells and uses the cellular transmembrane serine protease TMPRSS2 as a cooperating partner for S protein cleavage and priming.

Human ACE2 (angiotensin I converting enzyme (peptidyl-dipeptidase A) 2) 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.

CRISPR/Cas9-generated hACE2-hTMPRSS2-BALB/c mice combine the knock-in/knock-out mutations found in BALB/cJ-Ace2^{em2.1(ACE2)Synbl}/J (Stock No. 037077) and BALB/cJ-Tmprss2^{em2.1(TMPRSS2)Synbl}/J (Stock No. 037078). Expression of the mouse Ace2 (X-linked) and Tmprss2 genes is replaced with that of the homologous human genes. This strain has been useful in studies of Covid viral infection.

This strain has not yet been published (April, 2022). Please see the reference section below for any updates and further characterization information.

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.

Development

hACE2-hTMPRSS2-BALB/c mice combine the knock-in/knock-out mutations found in BALB/cJ-Ace2^{em2.1(ACE2)Synbl}/J (Stock No. 037077) and BALB/cJ-Tmprss2^{em2.1(TMPRSS2)Synbl}/J (Stock No. 037078). Expression of the mouse Ace2 and Tmprss2 genes is replaced with that of the homologous human genes.

Tmprss2^{em2.1(TMPRSS2)Synbl}:
Cas9 and gRNA Tmprss2-crRNA_5 (protospacer sequence 5’-GTGCTCAGACTGATACCCG-3’) were used to introduce the following at codon 19 in exon 3 of the mouse Tmprss2:

• GGSG linker: coding sequence for GGSG linker in-frame with upstream mouse Tmprss2 coding sequence.
• P2A: porcine teschovirus 2A self-cleaving peptide sequence.
• hTMPRSS2 CDS: human TMPRSS2 cDNA coding sequence with SV40 intron inserted for enhanced expression.
• hTMPRSS2 3’UTR: 1837 bp human TMPRSS2 3’UTR.
• hTMPRSS2 3’: 112 bp human intergenic sequence immediately 3’ of TMPRSS2 polyadenylation region.
• Tmprss2-gRNA5 Cassette: “Active genetics” cassette comprised of a mouse U6 promoter-driven Tmprss2-gRNA5 guide RNA with 2xPolIII terminator, all in reverse orientation relative to Tmprss2 gene.
• SA-SV40 poly(A) signal: splice acceptor and SV40 polyadenylation sequence designed to trap and truncate any mouse Tmprss2 isoforms that may splice past the hTMPRSS2 cassette.
• FRT-PGK-EM7-Neo-bGHpA-FRT: Neomycin resistance gene driven by the mouse PGK promoter and with a bovine growth hormone polyadenylation sequence. The FRT sites allow Flp-mediated removal of the neomycin cassette from targeted embryonic stem cell clones as well as collapse of targeted tandem integration events if required.

Ace2^{em2.1(ACE2)Synbl} (mutation is X-linked):
SpCas9 and gRNA Ace2-cRNA_32 (protospacer sequence 5’-AAGGCTGAGAAGGAGCCAGG-3’) were used to introduce the following 13 bp downstream of the mouse Ace2 start codon in exon 2:

• Mouse Signal Peptide: signal peptide coding sequence (translation produces amino acid sequence Ace2 WLLLSLVAVTTA) in-frame with first 5 amino acids of mouse Ace2
• hACE2 CDS: human ACE2 cDNA coding sequence with SV40 intron inserted for enhanced expression
• hACE2 3’UTR: 872 bp human ACE2 3’UTR
• hACE2 3’: 100 bp human intergenic sequence immediately 3’ of ACE2 polyadenylation region
• Ace2-gRNA32 Cassette: “Active genetics” cassette comprised of a mouse U6 promoter-driven Ace2-gRNA32 guide RNA with 2xPolIII terminator, all in reverse orientation relative to Ace2
• SA-SV40 poly(A) signal: splice acceptor and SV40 polyadenylation sequence designed to trap and truncate any mouse Ace2 isoforms that may splice past the hACE2 cassette.
• FRT-PGK-EM7-Neo-bGHpA-FRT: Neomycin resistance gene driven by the mouse PGK promoter and with a bovine growth hormone polyadenylation sequence. The FRT sites allow Flp-mediated removal of the neomycin cassette from targeted embryonic stem cell clones as well as collapse of targeted tandem integration events if required.

This strain was created and maintained on a BALB/cJ genetic background. The donating laboratory verified the correct integration and verified the lack of off-target backbone integration.

Upon arrival at The Jackson Laboratory, sperm was cryopreserved. Cryorecovery/live colony development will involve BALB/cJ (Stock No. 000651). (April, 2022)

Allele

Symbol: Ace2^{em2.1(ACE2)Synbl}
Name: endonuclease-mediated mutation 2.1, Synbal
MGI accession ID: MGI:7261383
Generation method: Endonuclease-mediated
Attributes: Null/Knockout; Inserted expressed sequence; Humanized sequence
Marker symbol: Ace2
Marker name: angiotensin I converting enzyme (peptidyl-dipeptidase A) 2
Marker synonyms: 2010305L05Rik; ACEH
Chromosome: X
Strain of origin: BALB/cJ
Molecular note: SpCas9 and gRNA Ace2-cRNA_32 (protospacer sequence 5’-AAGGCTGAGAAGGAGCCAGG-3’) are used to introduce the following sequence 13 bp downstream of the mouse Ace2 start codon in exon 2: signal peptide coding sequence (translation produces amino acid sequence Ace2 WLLLSLVAVTTA) in-frame with first 5 amino acids of mouse Ace2, human ACE2 cDNA coding sequence with SV40 intron, 872 bp human ACE2 3’UTR, 100 bp human intergenic sequence immediately 3’ of ACE2 polyadenylation region, cassette comprised of a mouse U6 promoter-driven Ace2-gRNA32 guide RNA with 2xPolIII terminator, all in reverse orientation relative to Ace2, splice acceptor and SV40 polyadenylation sequence designed to trap and truncate any mouse Ace2 isoforms that may splice past the hACE2 cassette, and a FRT-flanked PGK neomycin cassette with bGHpA. The FRT sites allow Flp-mediated removal of the neomycin cassette from targeted embryonic stem cell clones as well as collapse of targeted tandem integration events if required. Flp-mediated recombination removed the FRT-flanked neo cassette.

Allele

Symbol: Tmprss2^{em2.1(TMPRSS2)Synbl}
Name: endonuclease-mediated mutation 2.1, Synbal
MGI accession ID: MGI:7261385
Generation method: Endonuclease-mediated
Attributes: Null/Knockout; Inserted expressed sequence; Humanized sequence
Marker symbol: Tmprss2
Marker name: transmembrane protease, serine 2
Marker synonyms: D16Ertd61e; epitheliasin; PRSS10
Chromosome: 16
Strain of origin: BALB/cJ
Molecular note: Cas9 and gRNA Tmprss2-crRNA_5 (protospacer sequence 5’-GTGCTCAGACTGATACCCG-3’) are used to introduce the following sequences at codon 19 in exon 3 of the mouse Tmprss2 gene: GGSG linker, P2A, human TMPRSS2 cDNA coding sequence with SV40, 1837 bp human TMPRSS2 3’UTR, 112 bp human intergenic sequence immediately 3’ of TMPRSS2 polyadenylation region, cassette comprised of a mouse U6 promoter-driven Tmprss2-gRNA5 guide RNA with 2xPolIII terminator, all in reverse orientation relative to Tmprss2 gene, splice acceptor and SV40 polyadenylation sequence designed to trap and truncate any mouse Tmprss2 isoforms that may splice past the hTMPRSS2 cassette. FRT-flanked PGK neomycin cassette with a bGHpA. The FRT sites allow Flp-mediated removal of the neomycin cassette from targeted embryonic stem cell clones as well as collapse of targeted tandem integration events if required. Flp-mediated recombination removed the FRT-flanked neo cassette.