Genetically modified mice that serve as models of inherited diseases or as tools to dissect biological mechanisms have proven essential to the progress of biomedical science. Mice, like humans, are heavily colonized by micro-organisms that can be pathogenic, innocuous, or beneficial commensals. In humans and mice, the microflora roughly represents 90% of the cells that compose an individual. The phenotype associated with a specific mouse genome modification may result from disruption of the interactions between the host and its microflora. Commonly, mice are raised in the now standard Specific Pathogen Free (SPF) conditions, which do not significantly modify the extreme diversity and number of colonizing micro-organisms. Gnotobiology techniques allow for the generation and maintenance of animals in a germ-free or axenic environment and the possibility to restore specific components of the microflora. The comparative analysis of a given mouse line raised in SPF and in germ-free condition, reveals the contribution of the microflora to the phenotype associated with a specific mouse genotype. This approach has been essential e.g., to discriminate between autoimmunity and inflammatory immunopathologies in various mouse models. Germ-free animals can also help us to understand the host-commensal interactions during tissue regeneration, like in the intestinal epithelium. Metabolic disorders are now also clearly associated with the microbiota composition. The notion that the microbiota could also influence behaviour is being investigated as well. Finally, the importance of microbes in cancer and cancer therapy has been increasingly claimed in the past few years. To test new experimental settings on this expanding research area, the use of germ-free/gnotobiotic technology constitutes an attractive tool.

Over the last 10 years, gnotobiology has experienced a comeback, likely because of the development of high-throughput analyses of the microbiome and the development of empirical system biology. Interestingly, while differences in phenotype severity, penetrance or incidence for the same mouse genotype have been long noted between institutions and largely understood as an influence of the micro-environment, the effort to define SPF conditions – by exclusion criteria – at an international level did not fully solve the discrepancies. As an example, the identification of Helicobacter hepaticus as an opportunist pathogen that affects several biological parameters and its subsequent inclusion in the SPF list, helped the field of intestinal inflammation. More recently, the identification of Segmented Filamentous Bacteria (SFB) in the Taconic but not the Jackson C57BL/6 colonies has finally elucidated most intriguing phenotypic variations across institutions, in WT and in specific mutant mice that were cross-colonized. These two examples highlight that only a radical approach, such as axenization and colonization with a defined flora will ensure both reproducibility of investigations and proper assessment of the host genotype contribution to a given phenotype. It is worth noting that a minimal commensal flora is already defined and routinely used in some mouse production centres. Yet, it is also clear that even this restricted commensal flora (5-8 species) modify their host biology. In essence, it is becoming more common and there is indeed a requirement to assess the contribution of the microbiome to specific phenotypes.

To aid in assessing the contribution of the microbiome to specific phenotypes, the INFRAFRONTIER axenic service was funded and initiated by the INFRAFRONTIER2020 project, a four year infrastructure project that started in January 2017 and was supported by the EU Research and Innovation Program Horizon2020. Beyond providing pilot axenic services the INFRAFRONTIER2020 project supported capacity development in gnotobiology, and the coordination and development of a comprehensive INFRAFRONTIER service portfolio to support microbiome research.

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