Gt(ROSA)26Sor^{em27(KRT18-ACE2)Mvw}

CRISPR/cas9 genome editing and RMCE (recombinase-mediated cassette exchange) are utilized to insert a human keratin 18 promoter directing expression of the full length human ACE2with a C-terminal FLAG epitope tag into a modified Gt(ROSA)26Sor (NSG.RosaBxb-GT/GA) locus. The NOD.Cg-Prkdc Il2rg/SzJ (NSG)-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). (J:322700)