Characterizing a Lethal CAG-ACE2 Transgenic Mouse Model for SARS-CoV-2 infection with Using Cas9-Enhanced Nanopore Sequencing

Abstract

The SARS-CoV-2 pandemic has underscored the necessity for functional transgenic animal models for testing. Mouse lines with overexpression of the human receptor ACE2 serve as the primary animal model to study COVID-19 infection. Overexpression ofACE2under a strong ubiquitous promoter facilitates convenient and sensitive testing of COVID-19 pathology. We performed pronuclear microinjections using a 5 kb CAG-ACE2 linear transgene construct and identified three founder lines with 140, 72, and 73 copies, respectively. Two of these lines were further analyzed forACE2expression profiles and sensitivity to SARS-CoV-2 infection. Both lines expressedACE2in all organs analyzed. Embryonic fibroblast cell lines derived from transgenic embryos demonstrated severe cytopathic effects following infection, even at low doses of SARS-CoV-2 (0,1-1.0 TCID50). Infected mice from the two lines began to show COVID-19 symptoms three days post-infection and succumbed between days 4 and 7. Histological examination of lung tissues from terminally ill mice revealed severe pathological alterations. To further characterize the integration site in one of the lines, we applied Nanopore sequencing combined with Cas9 enrichment to examine the internal transgene concatemer structure. Oxford Nanopore sequencing (ONT) is becoming the gold standard for transgene insert characterization, but it is relatively inefficient without targeted region enrichment. We digested genomic DNA with Cas9 and gRNA against theACE2transgene to create ends suitable for ONT adapter ligation. ONT data analysis revealed that most of the transgene copies were arranged in a head-to-tail configuration, with palindromic junctions being rare. We also detected occasional plasmid backbone fragments within the concatemer, likely co-purified during transgene gel extraction, which is a common occurrence in pronuclear microinjections.

A potent, broadly neutralizing human monoclonal antibody that efficiently protects hACE2-transgenic mice from infection with the Wuhan, BA.5, and XBB.1.5 SARS-CoV-2 variants

“…C57BL/6-Tg (CAG-ACE2)5Nrba/Icg mice were generated through a pronuclear injection of a genetic construct containing the cDNA of the human ACE2 gene under the control of a strong ubiquitous constitutive chimeric CAG promoter ( 34 ). The transcription of the hACE2 cassette was observed in all organs of these mice, and hACE2 protein was detected on the cell surface.…”

“…In these transgenic mice, signs of viral infection were observed in the lungs following intranasal infection. No signs of infection in the brain were observed when analyzing the histological sections ( 34 ). The mice were obtained from the SPF animal facility of the Institute of Cytology and Genetics SB RAS (Novosibirsk, Russia).…”