Mouse ENU Mutagenesis to Understand Immunity to Infection: Methods, Selected Examples, and Perspectives

Abstract

Infectious diseases are responsible for over 25% of deaths globally, but many more individuals are exposed to deadly pathogens. The outcome of infection results from a set of diverse factors including pathogen virulence factors, the environment, and the genetic make-up of the host. The completion of the human reference genome sequence in 2004 along with technological advances have tremendously accelerated and renovated the tools to study the genetic etiology of infectious diseases in humans and its best characterized mammalian model, the mouse. Advancements in mouse genomic resources have accelerated genome-wide functional approaches, such as gene-driven and phenotype-driven mutagenesis, bringing to the fore the use of mouse models that reproduce accurately many aspects of the pathogenesis of human infectious diseases. Treatment with the mutagen N-ethyl-N-nitrosourea (ENU) has become the most popular phenotype-driven approach. Our team and others have employed mouse ENU mutagenesis to identify host genes that directly impact susceptibility to pathogens of global significance. In this review, we first describe the strategies and tools used in mouse genetics to understand immunity to infection with special emphasis on chemical mutagenesis of the mouse germ-line together with current strategies to efficiently identify functional mutations using next generation sequencing. Then, we highlight illustrative examples of genes, proteins, and cellular signatures that have been revealed by ENU screens and have been shown to be involved in susceptibility or resistance to infectious diseases caused by parasites, bacteria, and viruses.

Genetic and behavioral characterization of a Kmt2d mouse mutant, a new model for Kabuki Syndrome

“…Even today, several tools are available to manipulate the mouse genome to obtain transgenic, knockout and knock‐in variants. Forward genetics is still important, as random mutagenesis can be used to gather new information on already known genes . Furthermore, a significant proportion of human diseases is caused by nucleotide variants in genes that affect their function or regulation rather than merely deactivating them .…”

“…Considering the above, the study of animal models obtained by mutagenesis can be used to elucidate the function of genes and characterize human genetic diseases . The induction strategy for point mutations by N‐ethyl‐N‐nitrosourea (ENU) together with consistent phenotypic selection can be a powerful tool to identify mutations responsible for complex phenotypes …”

“…Forward genetics is still important, as random mutagenesis can be used to gather new information on already known genes. 2 Furthermore, a significant proportion of human diseases is caused by nucleotide variants in genes that affect their function or regulation rather than merely deactivating them. 3 Considering the above, the study of animal models obtained by mutagenesis can be used to elucidate the function of genes and characterize human genetic diseases.…”

“…4 The induction strategy for point mutations by N-ethyl-N-nitrosourea (ENU) together with consistent phenotypic selection can be a powerful tool to identify mutations responsible for complex phenotypes. 2 ENU is still the most used chemical compound for mutagenesis in mouse. 1 One of the major bottlenecks for studying mutants induced by ENU is the identification of the mutation by genetic mapping followed by the sequencing (Sanger method) of one or more genes in the mapped region.…”

In vitro responses to known in vivo genotoxic agents in mouse germ cells

“…Alkylating agents affect the mammalian genome by forming DNA lesions, and thus causing base substitution mutations, or preventing DNA replication [Imai et al, ; van Boxtel et al, ]. However, ENU has been shown to be a potent alkylating agent inducing germ cell line mutation due to its strength and preferential activity in spermatogonial stem cells [Caignard et al, ]. It has also been shown that germ cells treated with 6‐MP have the greatest response in early meiotic spermatocytes [Generoso et al, ; Norgard et al, ].…”