c57bl/6ncrl (b6n) germ-free mice · research applications b6n germ-free mice may be used as embryo...
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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
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
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
germ-free status of the rederived B6N colonies.
askcharlesriver@criver.com • www.criver.com
Husbandry
An example of the plastic isolators in which the germ-free
mice are housed is shown in Figure 1. Before being used,
an isolator is tested for leaks, chemically sterilized with 2%
peracetic acid, and ventilated. Sterilization of the ventilated
isolator is confirmed by culturing swabs of surfaces,
caging, and supplies collected from the isolator over several
weeks.
Once in use, an isolator is kept sterile by being ventilated
with HEPA-filtered air under positive pressure. Supplies,
such as food, bedding, water, and caging, are autoclaved
in transfer cylinders. To assure sterilization, supplies are
arranged in cylinders following standard configurations
(Figure 2). Self-contained bioindicators containing heat-
stable bacterial spores (e.g., EZTest® Steam, Mesa Labs)
and temperature indicators are placed throughout each
autoclave run, including inside a test cylinder. Autoclave
printouts are examined to confirm that the appropriate
cycle was chosen and ran without error. Temperature
indicators are evaluated and the bioindicators, including
those retrieved from inside the test cylinder, are incubated
(at 55-60 oC). Cylinders are released for use only if all
bioindicators read negative.
Figure 1. Plastic isolator for germ-free husbandry
View of a drum filled with cages, the inside of the drum showing underlying perforations in the steel structure required for
sterilizing steam to reach internal materials during the autoclave cycle.
To antiseptically import the supplies, the cylinder is attached to the isolator port with a plastic sleeve (Figure 3); the supplies
are transferred into the isolator through a double-door lock system that has been disinfected by spraying with 2% peracetic
acid or a sterilizing level of chlorine dioxide (e.g., CLIDOX-S® diluted 1:3:1). The double-door lock system is also used to
transfer animals, samples, and other materials in and out of the isolator. All manipulations of mice and supplies inside the
isolator are through gloves and sleeves attached to the isolator walls.
Figure 2. Cylinder for autoclaving supplies
Figure 3. Autoclaved cylinder attached to isolator
C57BL/6NCrl (B6N) Germ-Free Mice
Shipping
To preserve their germ-free status during shipment,
mice are transferred from the isolator in which they are
housed through the disinfected double-door lock system
into cages within a germ-free shipper sterilized with
ethylene oxide gas. Upon receipt of a lightweight shipper,
the shipper should be attached to the port of the isolator
and the mice antiseptically transferred.
askcharlesriver@criver.com • www.criver.com
Microbiological MonitoringGerm-free colonies are monitored for extraneous bacteria
and fungi, and for pathogens. Table 2 shows the test
methods, samples, and frequencies. Testing for extraneous
microbes is conducted frequently, based on the potentially
high incidence of this type of contamination and its
significant consequences to customers. Weekly, a slurry
from each isolator (consisting of feces and environmental
swabs in animal drinking water) is inoculated onto various
culture media, then incubated aerobically and anaerobically
in a dedicated anaerobic workstation. Because microbial
contaminants may be fastidious or non-cultivable on
cell-free media (like much of the indigenous microbiota),
culture-independent methods are employed. Wet mounts
of the slurries collected each week are examined by
phase microscopy for motile organisms. In addition, feces
collected quarterly from mice in each isolator are assayed
by PCR for the bacterial 16S ribosomal RNA (rRNA) gene.
Table 2. Microbiological Surveillance of Germ-Free Mouse Isolators
Methodology
Sample Type: Tests Frequency:
Slurry Weekly
Feces Quarterly
Animals Annually
MicrobiologyCulture Phase microscopy
X X
X X
PCRBacterial 16S rRNA Rodent Pathogens
X X
Gross and Microscopic ExamsNecropsy Parasitology
X X
Serology MFIA/IFA X
Comprehensive health monitoring for pathogens is
performed annually on mice from each isolator. This
comparatively low testing frequency is justified by the
historically negligible incidence of pathogens infecting
isolator-housed colonies. Animal organs and tissues
are grossly examined, and histopathology is carried out
if lesions suggesting an infectious disease process are
observed. Specimens from the gut and skin are examined
microscopically for endo- and ectoparasites. Blood
samples are screened for pathogen-specific antibodies by
the multiplexed fluorometric immunoassay (MFIA®), with
corroboration of unexpected (or indeterminate) findings
mostly by indirect immunofluorescence assays (IFA).
For cultural isolation of bacteria and fungi, various
microbiologic culture media are inoculated with
respiratory and gut samples and incubated aerobically
and anaerobically. Samples for anaerobic culture are
collected from euthanized mice dissected in an anaerobic
workstation. Isolates are identified both according to their
colonial and cellular morphology and by MALDI-TOF mass
spectrometry, and, if necessary, by PCR. In addition, swabs
of the skin, oral cavity, and feces are tested by PCR for
pathogens of all types.
The confirmed detection of bacterial, viral, parasitic, or
fungal agents in germ-free mice or isolators would result
in immediate cessation of shipment from the isolator and
immediate elimination of the isolator colony. Charles River
considers each isolator to be a microbiologic unit and
will not test and cull individual cages within an isolator.
It remains our policy to inform our customers in a timely
manner of any breaches in animal health or genetic integrity,
providing urgent colony health information via email or other
method. For assistance regarding specific information on
Charles River monitoring procedures, additional data on
animals, or interpretation of the monitoring information,
please direct inquiries to Charles River Technical Services
(877-274-8371) or email AskCharlesRiver@crl.com.
askcharlesriver@criver.com • www.criver.com © 2018, Charles River Laboratories International, Inc.
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