动物造模,转基因小鼠模型 z-bio 2022-10-20 384

　　Objective To obtain a humanized mouse model of hACE2 containing the full length hACE2 gene sequence, and to provide an important tool for the research of cardiovascular disease, pathogenesis of COVID-19, and drug and vaccine development.

　　Methods BAC plasmid containing hACE2 complete coding sequence was purified, and several first construction mice were obtained by microinjection of mouse fertilized eggs and oviduct transplantation. The lines were bred and established respectively. Stable F2 generation mouse strains were obtained through cross breeding. The hACE2 gene integration, gene copy number, relative fluorescence quantitative PCR, Western Blot and immunofluorescence of F2 generation mice were further analyzed.

　　Results Four mouse strains including hACE2-6-9, hACE2-14-3, hACE2-15-1 and hACE2-15-2 were obtained. Gene integration results showed that hACE2-6-9, hACE2-15-1 and hACE2-15-2 mouse strains contained complete hACE2 gene loci and long gene regulatory regions, and hACE2-14-3 contained complete hACE2 gene loci and short 3'UTR gene regulatory regions. The results of relative fluorescence quantitative PCR and Western Blot showed that the expression levels of hACE2 mRNA and protein in the intestine of hACE2-6-9, hACE2-15-1 and hACE2-15-2 strains were low, while the expression levels of hACE2-14-3 strains with shorter regulatory sequences were high. Immunofluorescence staining results showed that hACE2-15-1 strain mice had hACE2 expression in renal tubules and pulmonary vascular endothelial cells.

　　Conclusion The hACE2 humanized BAC transgenic mice containing the full length gene sequence have been obtained, and the complete hACE2 promoter and gene regulatory region have been retained, thus providing an important tool for the research on the pathogenesis of cardiovascular disease, COVID-19, the regulation mechanism of hACE2 gene expression, and the research and development of drugs and vaccines.