Inbred mice are critical tools in biomedical research and one particular strain, known as C57BL/6 (Black 6 or B6, for short), is rapidly becoming the standard model for many applications and revolutionising modern research.

Understanding the B6 mouse, its origins, characteristics and potential is required to make full and appropriate use of this model in research and drug discovery.

The B6 mouse is a versatile and powerful tool in preclinical research. These mice form the basis for many research models, including those in the fields of metabolic disease, obesity, diabetes, immune disorders, immuno-oncology and neurobiology, among others. No single animal model is as versatile as the B6 mouse, and its power is enhanced by the rich diversity of genetically-engineered models (GEMs) on the B6 background.

These combined properties make the B6 a vital tool for research efforts throughout the spectrum of drug discovery and development.

Rise in B6 usage

The B6 is now the most popular and significant mouse in research. Since 1990, the use of B6 mice has grown dramatically, and annual B6 citations in the scientific literature have risen by more than 800% during this period, according to Google Scholar. While this increase is partially driven by publication trends and increased biomedical funding, there has been a substantial shift toward using B6 mice versus other mouse strains. Out of the over 450 inbred mouse strains that have been developed, B6 now accounts for more than 60% of all published mouse studies, as compared to only 36% in 1990.

In response to rising demand for this valuable model, numerous vendors throughout the world now produce and distribute B6 mice for pharmaceutical, biotech and academic research. There are at least 18 different types of B6 mice (known as B6 substrains) that are commercially available. These B6 substrains are all distantly related and, although similar, can differ in several important aspects. Understanding the origins and diversity of these substrains is important in choosing an appropriate B6 substrain for a successful research project.

Origins and diversity of B6 mice

The history of the B6 dates to the 1920s and 1930s, when researchers sought a model in which all individual mice were genetically identical. To accomplish this, the B6 strain was created by extensive inbreeding over many generations. Every modern B6 mouse is a direct descendant of the one original B6 line; however, the family has grown significantly and there is no longer a single B6 strain, but rather numerous B6 substrains, each related but with different characteristics and suitability for research studies.

These modern B6 substrains are each genetically different and substrains should be considered distinct. In fact, the terms ‘B6’ or ‘C57BL/6’ are so ambiguous that their use is unadvisable as there has not been a single C57BL/6 strain for almost a century.

What is the right B6?

There are two major types of B6 mice: C57BL/6J (B6J) and C57BL/6N (B6N). The B6J substrains are all descendants from B6 mice originally maintained at The Jackson Laboratory, whereas the B6N substrains originate from a colony established in 1951 at the National Institutes of Health using B6 mice from The Jackson Laboratory. Since 1951, these two major branches of the B6 family have diverged, each accumulating numerous genetic differences, as explained later. This B6J/B6N divide remains the greatest source of variation between the modern B6 substrains; however, there are also substantial differences between substrains of the B6J or B6N varieties.

Today vendors produce genetically-defined B6J and B6N substrains that are each distinct, and specific vendor codes are used to differentiate these (eg, Tac for Taconic, J for The Jackson Laboratory, Crl for Charles River and Hsd for Envigo). In some cases, several vendor codes are used for strains maintained sequentially by different vendors (eg C57BL/6NJ or C57BL/6JBomTac). When using B6 mice it is very important to reference the full nomenclature that indicates its provenance and vendor code (eg, C57BL/6NTac from Taconic and C57BL/6J from The Jackson Laboratory).

The differences between B6 substrains is more than just semantics; not only are they genetically unique, but these attributes also affect their suitability for use as specific research models. In general, B6N substrains are recommended for studies addressing diabetes, metabolism, obesity and immunology, whereas B6J substrains may be more appropriate for certain behavioural assays. In some cases, potential genetic causes for these differences have been identified. Most B6J substrains have defective mitochondria due to a mutation in the Nnt gene, which may explain why B6J do not perform as well as B6N in diabetes and obesity models.

Furthermore, all B6J substrains have a mutation in an important immune system gene, Nlrp12, which may affect their suitability as models for immunological disorders. In contrast, all B6N mice develop mild vision problems with age (Crb1 mutation) and have dysfunctional brain reward circuitry (Cyfip2 mutation), making B6N mice less appropriate for certain models of neuropathology or addiction.

In addition to these major differences between the B6J and B6N substrains, individual substrains may possess other mutations that affect their performance. One example of a B6J strain that differs from others is C57BL/6JOlaHsd, which has a deletion of genes involved in neurodevelopment (Snca) and blood clotting (Mmrn1). Among the B6N, the C57BL/6NHsd substrain has impaired immune system function due to a Dock2 mutation.

With such extensive variation between different B6 substrains, it is obvious that selecting a particular substrain for use should be based upon scientific justification and performance for the model in question above any other consideration. Although all B6 mice look very similar, they are far from carbon copies, and the B6 substrains produced and sold by different vendors can and do perform very differently. The choice and source of B6 substrains is critically important and should be a top priority when establishing and validating research models.

The microbiome and B6 model performance

While genetic variation between B6 substrains has been acknowledged for some time, there is a growing appreciation for how the microbiome affects the performance of B6 models. The microbiome encompasses the entire population of all microorganisms (bacteria, fungi, etc) that inhabit the body, and modern research has shown that the microbiome can have major impacts on health and disease.

Differences in the microbiome in B6 mice can have an equal, if not greater, impact on model performance as compared to genetic influences. The effect of the microbiome has been shown in a wide variety of research models, including infectious disease, immunological disorders, oncology, diabetes (Type 1 and Type 2), obesity and neurologic disorders (eg, autism, Parkinson’s and depression), to name a few.

The microbiome is also suspected to be one of the most impactful variables contributing to irreproducibility in animal models. Unfortunately, in this relatively new field there are no standard approaches to address the microbiome in B6 models and relatively few options available for researchers to control for its effects or harness its potential.

The animal vendor industry does not typically provide options to buy B6 mice with a known microbiome, but rather relies on the exclusion of specific pathogenic organisms. While excluding a pathogen can be straightforward, controlling the overall microbiome of a mouse is challenging. The microbiome of a B6 mouse is determined by a number of key factors: environmental exposure (from housing location and other mice), husbandry conditions, diet and water.

Typically, B6 mice produced in a single location will tend to have similar microbiomes, compared to those produced in other locations, even if all other factors are tightly controlled. For example, B6 mice ordered from different vendors, and even different production locations from the same vendor, can possess very different microbiomes. A general recommendation is to obtain B6 mice from specific locations (ie, barrier facilities) when validating a model to select for a microbiome profile that is optimal and then to exclusively source from that location throughout the life of the study.

Although there are few options for researchers to select a specific B6 microbiome, one option is to select mice that carry specific commensal (ie, nonpathogenic) organisms that are known to affect model performance. One such example is an organism known as segmented filamentous bacteria (SFB). SFB lives in the mouse gut, where it regulates immune system development, and has been shown to affect the performance of immunology, diabetes and obesity models.

Only one vendor, Taconic Biosciences, reports on the presence or absence of this important organism and provides options to obtain B6 mice with or without SFB. Other strategies to control the microbiome in B6 models are still in their infancy, but the recognition of this important factor will hopefully lead to better microbiome options for researchers.

Considerations for selecting B6 mice

Selecting an appropriate B6 and using it to its fullest potential is challenging, yet critical for the successful completion of any research project.

Unlike many other tools and consumables in the biomedical industry, there is no standard B6 and there are few established criteria to monitor performance. A pipette, for instance, will either deliver liquids with accuracy and precision, or it will not. Either way, this can be determined empirically, and there are set criteria for evaluating one pipette versus another. As another example, chemicals are sold as known molecular entities at specified levels of purity. There is a reasonable expectation that one chemical compound will work just as well as another, given that same level of purity.

In contrast, the biological basis of B6 mice is complex and messy. Unlike the pipette, there is no easy way to establish equivalency between different B6 mice. Unlike the purity of a chemical compound, there is no way to know and quantify all the genetic, environmental and microbiome factors that would affect model performance. Instead, B6 models should be selected for a given application based on validation studies that consider all the variables that affect performance, including substrain, vendor source and microbiome.

The B6 toolbox: reshaping possibilities, enabling discovery

Other than humans, more is known about the B6 genome than any other mammal. The B6 genome was first sequenced in 2002, and this knowledge has helped the B6 become the standard strain not only for research models but also for the creation of GEMs. Combined with its versatility and multiple applications, the B6 is now preferred for most GEM projects.

One particular B6 substrain, C57BL/6NTac, was chosen by international consortia to be the source of embryonic stem (ES) cells used for all new GEMs. As a result, more than 22,000 mouse knockout lines have been created on this C57BL/6NTac background. In contrast to older techniques, which relied on other, less-useful mouse strains, the ability to generate GEMs directly on a B6 background accelerates timelines and increases research possibilities. Together with CRISPR gene editing techniques, B6 ES cells make it possible to generate research models with ever increasingly complex genetic alterations, such as mice-possessing humanised genes for testing biologics.

This rich resource of GEMs on a defined background is reshaping what is possible for researchers, by allowing them to understand how specific genes contribute to numerous disease processes and to identify novel therapeutic targets. The fact that all these mice are on the B6 background is important for several reasons.

– First, it enables direct comparisons between different GEMs since only the engineered genes differ between these mice and wildtype B6 controls.

– Second, since their genetic background is the same, this makes it possible to perform complex experiments involving genetic crosses and/or cell/tissue transplants between different GEMs.

– Thirdly, and perhaps most importantly, a GEM on a B6 background can be used in all the same diverse experimental models as wildtype B6 mice.

Of course, there are several caveats to these approaches, most importantly that the genetic background of the GEMs in question are on a defined B6 substrain with equivalent microbiomes to exclude the confounding effects of these variables, as discussed earlier. Nevertheless, this toolbox of B6 GEMs, along with the B6’s research versatility, combine to provide a powerful platform for research and discovery.

Advanced applications for B6 mice

The obese B6 – B6 mice, especially B6N substrains, are exceptionally prone to developing obesity, insulin resistance and diabetes when fed a high-fat diet. This makes the obese B6 useful in identifying therapeutic options for the modern obesity epidemic and associated conditions such as diabetes, metabolic syndrome, non-alcoholic fatty liver disease and nonalcoholic steatohepatitis. Standard high-fat diets will reproducibly induce weight gain, insulin resistance and hyperglycemia in both wildtype B6 mice and GEMs on the B6 background. B6 GEMs are making it possible to identify new targets for obesity and diabetes and play an important role in validating therapeutic targets.

The B6 in immuno-oncology – Modern immuno-oncology research has seen a resurgence in the use of the B6 in the form of syngeneic tumour models. These models make it possible to study how the immune system interacts with a tumour in the context of a full complement of immune cells, which is important in identifying and validating new therapeutic targets for immuno-oncology. The B6 has one of the most fully characterised immune systems of any animal, and there is a wide range of analytical tools and genetic models available to interrogate its function. Humanised B6 GEMs make it possible to test human-specific biologic immunotherapies in a syngeneic context. Furthermore, there is a growing number of engineered tumours developed from tumour GEM models on the B6 background. Thus, the B6 provides one of the most powerful platforms for immuno-oncology discovery.

The B6 in microbiome discovery – One of the most important tools in studying the microbiome is the germ-free mouse, which lacks all micro-organisms and is maintained in sterile isolators. Germ-free mice are used to study the microbiome in depth and to discover how microbes affect health and disease. Although any mouse can be made germ-free, the germ-free B6 is rapidly starting to dominate this new microbiome research field. The microbiome is linked to many different diseases, such as immune disorders, obesity and even cancer, and the B6 mouse is one of the best models for studying these. Germ-free B6 mice are making it possible to understand how the microbiome contributes to these conditions and enabling the development of new therapeutic avenues targeting the microbiome.

Conclusions

The B6 is the most important animal model in modern research. Understanding the critical genetic and microbiome factors that affect B6 model performance will aid in the selection of B6 models and improve the success of a research programme. From humble beginnings, this little black mouse has been transformed into one of the most useful tools in biomedical research and is being engineered and employed in ever increasingly complex applications.

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This article originally featured in the DDW Fall 2017 Issue

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Dr Philip Dubé is an Applications Scientist at Taconic Biosciences. Dr Dubé has a PhD in Physiology and an Honor’s BSc in Pharmacology from the University of Toronto. He has more than 16 years of experience in the use of rodent models and provides a wide range of knowledge in the application and execution of successful research studies. He specialises in animal models for microbiome, inflammatory disorders, oncology, immuno-oncology and metabolic disease research; gnotobiotic and germ-free animal models, their care and use; genetics of rodent animal models; and general animal husbandry, care and use.