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Human ACE2 DNA consisting of 5' UTR, CDS (exons 2-19) fused to mouse Ace2 3'UTR replaced the DNA region in mouse Ace2 gene stretching from the 5'UTR to the starting ATG in exon 2. The approach minimizes potential downstream splicing or RNP re-cleavage. Expression of the human CDS is primarily controlled by the mouse regulatory system and is expected to not transcribe the mouse version. Gene expression showed expected human ACE2 levels whereas mouse Ace2 was not expressed. CRISPR RNP was used to assist with homologous recombination using dsDNA repair in JM8A3 ES cells.
Human TMPRSS2 exons 3-14 (ENSG00000184012.14) was inserted in-frame into exon 2 (ENSMUSE00000641779) of the mouse Tmprss2 gene. CRISPR RNP was used to assist with homologous recombination (HR) using dsDNA repair template in ES cells. The mouse exon 2 splice acceptor and the guide protospacer (nt 1-17) were removed from the HR template to prevent potential downstream splicing or RNP re-cleavage. The expression of the human CDS is controlled by the mouse regulatory systems (including 3’UTR), and predicted to terminate with the transgene’s STOP codon without expression of the endogenous sequence. Gene expression showed expected human TMPRSS2 levels whereas mouse Tmprss2 was not expressed. CRISPR RNP was used to assist with homologous recombination using dsDNA repair template in JM8A3 ES cells derived from C57BL/6N.
Human FURIN DNA consisting of CDS (exons 2-16) fused to mouse Furin 3'UTR replaced start codon in mouse Furin gene. This approach minimizes potential downstream splicing or RNP re-cleavage as the guide protospacer was split. Expression of the human CDS is primarily controlled by the mouse regulatory system and is expected to not transcribe the mouse version. CRISPR RNP was used to assist with homologous recombination using dsDNA repair in JM8A3 ES cells.
For each of the three lines above, following blastocyst injection, the chimeras were mated with C57BL/6NCrl to create germline heterozygotes. Long-range PCR products were fully sequenced; potential off-targets were absent. Subsequent backcross was to C57BL/6NCrl.
Conditional phenotype: No
Colony Surveillance Program and Current Health Reports
Limited quantities of breeder mice (up to 2 males and 2 females or 4 mice) per investigator per month are available from a live colony, usually available to ship in under 12 weeks. Larger quantities may be available, please contact the distributing center directly at mmrrc@ucdavis.edu for more details.
Cryopreserved material may be available upon request, please inquire to mmrrc@ucdavis.edu for more information.
The donor or their institution limits the distribution to non-profit institutions only.
Distribution of this strain requires submission of the MMRRC Conditions of Use (COU). A link to the COU web form will be provided via email after an order has been placed; the form should be completed then or the email forwarded to your institutional official for completion.
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