www.criver.com EVERY STEP OF THE WAY C57BL/6NCrl (B6N) Germ-Free Mice Background Soon after birth, the gastrointestinal tract and other body surfaces of mammals are colonized by complex communities of microorganisms, traditionally termed microflora; more recently, they have also been called microbiota and microbiomes, which some differentiate as referring to microbial taxa and genomes, respectively. The “normal” autochthonous (i.e., indigenous) mammalian gut microbiota consist largely of beneficial, or commensal, bacteria that synthesize vitamins essential to host nutrition and provide a barrier to infection by pathogens. Gut flora also include significant numbers of archaea, eukaryotes, and viruses (including bacteriophages). 1 Microbes are by far most numerous in the large intestines, with concentrations that can reach trillions of microbial cells per gram of feces in the colon and represent 1,000 different species. 2 In humans, the number of cells that compose the microbiota reportedly are equivalent to or 10-fold greater than the number of human somatic cells, depending on whether nonnucleated erythrocytes are counted. 3 Therefore, it is not surprising that the gut and other microbiota have been found to play a key role in the development and homeostasis of host anatomy, physiology, metabolism, and immunity, as evidenced by the many abnormalities, such as an underdeveloped immune system and a markedly enlarged cecum, that characterize axenic (germ-free) rodents demonstrably free of all foreign bacteria as well as fungi, protozoa, parasites, and viruses. 4 Research into the role of microbiota in health and disease has increased exponentially during the past decade, encouraged by advances in molecular genetics that have led to the development of numerous genetically engineered mutant animal models, as well as sophisticated, culture-independent molecular tools for analyzing the microbiome, notably massively parallel “next-generation” DNA sequencing. 5 This research has demonstrated that the constituents of the gut microflora can abrogate or accentuate the phenotypes of mutant models. 6,7,8 Clinical studies have linked dysbiosis, or imbalances of microbiota, and the loss of microbial diversity (in part caused by the overuse of antibiotics in agriculture and medicine) to spikes in the incidence of an array of human diseases, ranging from juvenile diabetes to autism. 9,10 RESEARCH MODELS AND SERVICES Summary Germ-free rodents have been essential to microbiome research and the production of specific pathogen-free (SPF) rodent models. This document describes the background, uses, production, shipment, and microbiological monitoring of Charles River’s C57BL/6NCrl (B6N) germ-free mice.
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C57BL/6NCrl (B6N) Germ-Free Mice · Research Applications B6N germ-free mice may be used as embryo transfer recipients or foster dams for germ-free rederivation of mutant mouse models.
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www.criver.com
EVERY STEP OF THE WAY
C57BL/6NCrl (B6N) Germ-Free MiceBackgroundSoon after birth, the gastrointestinal tract and other
body surfaces of mammals are colonized by complex
communities of microorganisms, traditionally termed
microflora; more recently, they have also been called
microbiota and microbiomes, which some differentiate as
referring to microbial taxa and genomes, respectively.
The “normal” autochthonous (i.e., indigenous) mammalian
gut microbiota consist largely of beneficial, or commensal,
bacteria that synthesize vitamins essential to host nutrition
and provide a barrier to infection by pathogens. Gut flora
also include significant numbers of archaea, eukaryotes,
and viruses (including bacteriophages).1 Microbes
are by far most numerous in the large intestines, with
concentrations that can reach trillions of microbial cells per
gram of feces in the colon and represent 1,000 different
species.2
In humans, the number of cells that compose the
microbiota reportedly are equivalent to or 10-fold greater
than the number of human somatic cells, depending on
whether nonnucleated erythrocytes are counted.3 Therefore,
it is not surprising that the gut and other microbiota have
been found to play a key role in the development and
homeostasis of host anatomy, physiology, metabolism,
and immunity, as evidenced by the many abnormalities,
such as an underdeveloped immune system and a markedly
enlarged cecum, that characterize axenic (germ-free)
rodents demonstrably free of all foreign bacteria as well as
fungi, protozoa, parasites, and viruses.4
Research into the role of microbiota in health and disease
has increased exponentially during the past decade,
encouraged by advances in molecular genetics that
have led to the development of numerous genetically
engineered mutant animal models, as well as sophisticated,
culture-independent molecular tools for analyzing the
that the constituents of the gut microflora can abrogate
or accentuate the phenotypes of mutant models.6,7,8
Clinical studies have linked dysbiosis, or imbalances of
microbiota, and the loss of microbial diversity (in part
caused by the overuse of antibiotics in agriculture and
medicine) to spikes in the incidence of an array of human
diseases, ranging from juvenile diabetes to autism.9,10
RESEARCH MODELS AND SERVICES
SummaryGerm-free rodents have been
essential to microbiome research
and the production of specific
pathogen-free (SPF) rodent models.
This document describes the
background, uses, production,
shipment, and microbiological
monitoring of Charles River’s
C57BL/6NCrl (B6N) germ-free mice.
C57BL/6NCrl (B6N) Germ-Free Mice
Furthermore, the composition of patients’ microflora has
recently been reported to influence the efficacy of cancer
immunotherapy.11,12 Thus, studying and explicating the
interaction between hosts and their microbiota is of critical
importance to public health as well as animal research.
Charles River’s experience with germ-free technology
goes back to the 1950s, when the veterinarian who
founded Charles River Laboratories, Dr. Henry Foster, and
his colleagues incorporated germ-free rederivation into
the “cesarean-originated barrier-sustained” process they
pioneered for the large-scale production of SPF mice and
rats.13 In this process, germ-free rodents are associated
(i.e., colonized) with a defined cocktail of commensal
bacteria to normalize their physiology and prime their
immune systems. The cocktail most often used for this
purpose is the altered Schaedler flora (ASF) developed by
Roger Orcutt and colleagues at Charles River in the 1970s
(Table 1).14,15 In contrast to the original Schaedler flora16 on
which it was based, the ASF is fully anaerobic; moreover,
half of the eight species of bacteria in the ASF are extremely
oxygen-sensitive (EOS) fusiform anaerobes highly
representative of the autochthonous microbiota. Germ-
free and defined flora-associated animals are classified as
gnotobiotic, from the Greek roots gnostos (“known”) and
bios (“life”). By contrast, barrier-maintained SPF rodents
develop a complex microbiota that is defined only to the
extent that it does not include a limited list of pathogens.
Table 1. Compositive of Charles River Altered Schaedler Flora (ASF)*
Designation In Original Schaedler Taxonomy Genbank Accession
ASF 356 X Clostridium species AQFQ00000000.1
ASF 360 X Lactobacillus intestinalis AQFR00000000.1
ASF 361 X Lactobacillus murinus AQFs00000000.1
ASF 457 Mucispirillum schaedleri AYGZ00000000.1
ASF 492 Eubacterium plexicaudatum AQFT00000000.1
ASF 500 Pseudoflavonifactor species AYJP00000000.1
ASF 502 Clostridium species AQFU00000000.1
ASF 519 X Parabacteroides goldsteinii AQFV00000000.1
* The four ASF bacteria from the original Schaedler flora were isolated from the stomach and intestines of NCS mice in the 1960s by Russell W. Schaedler at Rockefeller University. The other ASF organisms were originally isolated from the large intestine of CD-1 mice in the 1960s at Charles River by Roger P. Orcutt (a graduate student of Schaedler’s).
Research ApplicationsB6N germ-free mice may be used as embryo transfer
recipients or foster dams for germ-free rederivation of
mutant mouse models. In addition, they may be compared
to SPF or Elite (opportunistic pathogen-free) B6N mice
to generally assess the relationship between microbiota
and phenotypes. Alternatively, the germ-free B6N mice
may be associated with a single microbial species (mono-
associated), defined microbiota like the ASF, or complex
polymicrobial mixtures to measure and understand the
effects of microbiota on phenotypes and experimental
responses.17,18,19 Germ-free mice have also been engrafted
with human microbiota by fecal transfer or inoculation of
defined microflora in order to investigate the contribution of
microbe-host relationships to human diseases.20
Production
Rederivation
The B6N strain was obtained by Charles River from the
National Institutes of Health in 1974. The current colonies of
germ-free B6N mice were rederived by sterile hysterectomy
followed by fostering on germ-free dams provided by the
Gnotobiotics and Microbiology Core at Boston Children’s
Hospital. Extensive testing by culture and culture-
independent methods described below has verified the
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