Characterizing a lethal CAG-ACE2 transgenic mouse model for SARS-CoV-2 infection using Cas9-enhanced nanopore...

Area of Science:

• * Molecular Biology and Genetics
• * Virology and Immunology
• * Animal Models in Disease Research

Background:

• * The COVID-19 pandemic necessitates robust animal models for therapeutic development.
• * Transgenic mice overexpressing the human Angiotensin-Converting Enzyme 2 (ACE2) receptor are critical for studying SARS-CoV-2 (the virus that causes COVID-19) infection.
• * Efficient methods for generating and characterizing these models are essential for research.

Purpose of the Study:

• * To generate and characterize transgenic mouse lines overexpressing human ACE2 for COVID-19 research.
• * To assess the susceptibility and pathological outcomes of SARS-CoV-2 infection in these novel mouse models.
• * To analyze the transgene integration site and concatemer structure using advanced sequencing techniques.

Main Methods:

• * Pronuclear microinjection of a CAG-ACE2 transgene construct into mouse embryos.
• * SARS-CoV-2 infection of transgenic mice and derived cell lines.
• * Analysis of ACE2 expression, cytopathic effects, clinical signs, and histopathology.
• * Nanopore sequencing with Cas9 enrichment for transgene integration site and structure analysis.

Main Results:

• * Successfully generated three founder transgenic mouse lines with high copy numbers of the CAG-ACE2 transgene.
• * Two lines exhibited widespread ACE2 expression and severe cytopathic effects in embryonic fibroblasts upon SARS-CoV-2 exposure.
• * Infected mice displayed rapid onset of COVID-19 clinical signs and mortality, with significant lung pathology.
• * Nanopore sequencing revealed a predominantly head-to-tail concatemer arrangement of transgene copies, with rare plasmid backbone fragments.

Conclusions:

• * The developed transgenic mouse lines effectively model SARS-CoV-2 infection, exhibiting high susceptibility and pathology.
• * These models are valuable tools for preclinical testing of COVID-19 therapeutics and vaccines.
• * Advanced sequencing methods provide detailed insights into transgene integration and structure, crucial for model characterization.