Figure 1.

Construction and Characterization of hACE2 Humanized Mouse

(A) The hACE2 gene were inserted into exon2, the first coding exon, of mouse ACE2 locating in chromosome X GRC m38.p6.

(B) The mice produced both 4,188 bp and 4,405 bp PCR amplicons that were identified as founder. Ten representative positive PCR results were shown.

(C) F1⁺ mice were sequenced to confirm the right insertion. The head and tail bases of each part were aligned with expected one.

(D) Two endonucleases, NcoI and StuI, were used to digest DNA for identifying the right insertion and no random recombinant. The expected sizes of WT and gene-targeted bands were 5.6 kb and 8.0 kb, respectively. Heterozygous female mice had both bands, whereas homozygous male mice had only one 8.0 kb band. All the mice detected with WPRE probe had only one 12.4 kb band, indicating no random insertion.

(E) western blotting results of tissue distribution of hACE2 in humanized mice and WT mice. The primary antibody used here cross-reacted with both mACE2 and hACE2.

(F and G) The expression pattern of mACE2 (F) and hACE2 (G) in hACE2 heterozygous and homozygous and WT mice (n = 3) detected by real-time qPCR.

(H) Bioluminescence imaging (BLI) of homozygous mice and WT mice.

(I) Immunofluorescence staining analysis for the hAEC2-expressed cell types in lung of hACE2 mice. Paraffin lung sections were stained for hACE2 (white), tdTomato (red) CC10 (green), β-IV-Tubulin (cyan), PDPN (magenta), and SPC (gold). Two white frames were magnified below. Solid red and yellow arrows indicated the ACE2⁺/tdTomato⁺/SPC⁺ cells and ACE2⁺/tdTomato⁺/CC10⁺ cells, respectively.