Mice Humanized for MHC and hACE2 with High Permissiveness to SARS-CoV-2 Omicron Replication.

Human Angiotensin-Converting Enzyme 2 (hACE2) is the major receptor enabling host cell invasion by SARS-CoV-2 via interaction with Spike. The murine ACE2 does not interact efficiently with SARS-CoV-2 Spike and therefore the laboratory mouse strains are not permissive to SARS-CoV-2 replication. Here, we generated new hACE2 transgenic mice, which harbor the hACE2 gene under the human keratin 18 promoter, in "HHD-DR1" background. HHD-DR1 mice are fully devoid of murine Major Histocompatibility Complex (MHC) molecules of class-I and -II and express only MHC molecules from Human Leukocyte Antigen (HLA) HLA 02.01, DRA01.01, DRB1.01.01 alleles, widely expressed in human populations. We selected three transgenic strains, with various hACE2 mRNA expression levels and distinctive profiles of lung and/or brain permissiveness to SARS-CoV-2 replication. These new hACE2 transgenic strains display high permissiveness to the replication of SARS-CoV-2 Omicron sub-variants, while the previously available B6.K18-ACE22Prlmn/JAX mice have been reported to be poorly susceptible to infection with Omicron. As a first application, one of these MHC- and ACE2-humanized strains was successfully used to show the efficacy of a lentiviral-based COVID-19 vaccine.

The human EF1a promoter does not provide expression of the transgene in mice.

In this work, we set out to create mice susceptible to the SARS-CoV-2 coronavirus. To ensure the ubiquitous expression of the human ACE2 gene we used the human EF1a promoter. Using pronuclear microinjection of the transgene construct, we obtained six founders with the insertion of the EF1a-hACE2 transgene, from which four independent mouse lines were established. Unfortunately, only one line had low levels of hACE2 expression in some organs. In addition, we did not detect the hACE2 protein in primary lung fibroblasts from any of the transgenic lines. Bisulfite sequencing analysis revealed that the EF1a promoter was hypermethylated in the genomes of transgenic animals. Extensive analysis of published works about transgenic animals indicated that EF1a transgenic constructs are frequently inactive. Thus, our case cautions against using the EF1a promoter to generate transgenic animals, as it is prone to epigenetic silencing.

Creation of transgenic mice susceptible to coronaviruses: a platform for studying viral pathogenesis and testing vaccines.

Over the past 20 years, coronaviruses have caused three epidemics: SARS-CoV, MERS-CoV, and SARS-CoV2, with the f irst two having a very high lethality of about 10 and 26 %, respectively. The last outbreak of coronavirus infection caused by SARS-CoV2 in 2019 in China has swept the entire planet and is still spreading. The source of these viruses in humans are animals: bats, Himalayan civets, and camels. The genomes of MERS-CoV, SARS-CoV and SARS-CoV2 are highly similar. It has been established that coronavirus infection (SARS-CoV and SARS-CoV2) occurs through the viral protein S interaction with the lung epithelium - angiotensin-converting enzyme receptor 2 (ACE2) - due to which the virus enters the cells. The most attractive model for studying the development of these diseases is a laboratory mouse, which, however, is resistant to coronavirus infection. The resistance is explained by the difference in the amino acid composition of mouse Ace2 and human ACE2 proteins. Therefore, to create mice susceptible to SARS- CoV and SARS-CoV2 coronaviruses, the human ACE2 gene is transferred into their genome. The exogenous DNA of the constructs is inserted into the recipient genome randomly and with a varying number of copies. Based on this technology, lines of transgenic mice susceptible to intranasal coronavirus infection have been created. In addition, the use of the technology of targeted genome modif ication using CRISPR/Cas9 made it possible to create lines of transgenic animals with the insertion of the human ACE2 gene under the control of the endogenous murine Ace2 gene promoter. This "humanization" of the Ace2 gene makes it possible to obtain animals susceptible to infection with coronaviruses. Thus, transgenic animals that simulate coronavirus infections and are potential platforms for testing vaccines have now been created.

Transgenic Model Systems Have Revolutionized the Study of Disease.

The current pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected most of the world in a profound way. As an indirect consequence, the general public has been put into direct contact with the research process, almost in real time. Justifiably, a lot of this focus has been targeted toward research directly linked to coronavirus disease 2019 (COVID-19). In this opinion article, we want to highlight to a general audience the value of having a diverse "portfolio" of research approaches for society as a whole. In this study, we will focus on our field of research, namely the study of gene regulation through the use of transgenesis. We will highlight how this type of research can also be used to provide a better understanding as well as tools to fight SARS-CoV-2 and other future challenges.