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C57BL/6Ncrl (B6N) Germ-Free Mice

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C57BL/6Ncrl (B6N) Germ-Free Mice 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. RESEARCH MODELS AND SERVICES C57BL/6NCrl (B6N) Germ-Free Mice Background been found to play a key role in the development and Soon after birth, the gastrointestinal tract and other homeostasis of host anatomy, physiology, metabolism, body surfaces of mammals are colonized by complex and immunity, as evidenced by the many abnormalities, communities of microorganisms, traditionally termed such as an underdeveloped immune system and a markedly microflora; more recently, they have also been called enlarged cecum, that characterize axenic (germ-free) microbiota and microbiomes, which some differentiate as rodents demonstrably free of all foreign bacteria as well as referring to microbial taxa and genomes, respectively. fungi, protozoa, parasites, and viruses.4 The “normal” autochthonous (i.e., indigenous) mammalian Research into the role of microbiota in health and disease gut microbiota consist largely of beneficial, or commensal, has increased exponentially during the past decade, bacteria that synthesize vitamins essential to host nutrition encouraged by advances in molecular genetics that and provide a barrier to infection by pathogens. Gut flora have led to the development of numerous genetically also include significant numbers of archaea, eukaryotes, engineered mutant animal models, as well as sophisticated, 1 and viruses (including bacteriophages). Microbes culture-independent molecular tools for analyzing the are by far most numerous in the large intestines, with microbiome, notably massively parallel “next-generation” concentrations that can reach trillions of microbial cells per DNA sequencing.5 This research has demonstrated gram of feces in the colon and represent 1,000 different that the constituents of the gut microflora can abrogate 2 species. or accentuate the phenotypes of mutant models.6,7,8 In humans, the number of cells that compose the Clinical studies have linked dysbiosis, or imbalances of microbiota reportedly are equivalent to or 10-fold greater microbiota, and the loss of microbial diversity (in part than the number of human somatic cells, depending on caused by the overuse of antibiotics in agriculture and whether nonnucleated erythrocytes are counted.3 Therefore, medicine) to spikes in the incidence of an array of human it is not surprising that the gut and other microbiota have diseases, ranging from juvenile diabetes to autism.9,10 EVERY STEP OF THE WAY www.criver.com Furthermore, the composition of patients’ microflora has immune systems. The cocktail most often used for this recently been reported to influence the efficacy of cancer purpose is the altered Schaedler flora (ASF) developed by immunotherapy.11,12 Thus, studying and explicating the Roger Orcutt and colleagues at Charles River in the 1970s interaction between hosts and their microbiota is of critical (Table 1).14,15 In contrast to the original Schaedler flora16 on importance to public health as well as animal research. which it was based, the ASF is fully anaerobic; moreover, Charles River’s experience with germ-free technology half of the eight species of bacteria in the ASF are extremely goes back to the 1950s, when the veterinarian who oxygen-sensitive (EOS) fusiform anaerobes highly founded Charles River Laboratories, Dr. Henry Foster, and representative of the autochthonous microbiota. Germ- his colleagues incorporated germ-free rederivation into free and defined flora-associated animals are classified as the “cesarean-originated barrier-sustained” process they gnotobiotic, from the Greek roots gnostos (“known”) and pioneered for the large-scale production of SPF mice and bios (“life”). By contrast, barrier-maintained SPF rodents rats.13 In this process, germ-free rodents are associated develop a complex microbiota that is defined only to the (i.e., colonized) with a defined cocktail of commensal extent that it does not include a limited list of pathogens. bacteria to normalize their physiology and prime their 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 Applications Production B6N germ-free mice may be used as embryo transfer Rederivation recipients or foster dams for germ-free rederivation of The B6N strain was obtained by Charles River from the mutant mouse models. In addition, they may be compared National Institutes of Health in 1974. The current colonies of to SPF or Elite (opportunistic pathogen-free) B6N mice germ-free B6N mice were rederived by sterile hysterectomy to generally assess the relationship between microbiota followed by fostering on germ-free dams provided by the and phenotypes. Alternatively, the germ-free B6N mice Gnotobiotics and Microbiology Core at Boston Children’s may be associated with a single microbial species (mono- Hospital. Extensive testing by culture and culture- associated), defined microbiota like the ASF, or complex independent methods described below has verified the polymicrobial mixtures to measure and understand the germ-free status of the rederived B6N colonies. 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 C57BL/6NCrl (B6N) Germ-Free Mice Husbandry An example of the plastic isolators in which the germ-free isolator is confirmed by culturing swabs of surfaces, mice are housed is shown in Figure 1. Before being used, caging, and supplies collected from the isolator over several an isolator is tested for leaks, chemically sterilized with 2% weeks. peracetic acid, and ventilated. Sterilization of the ventilated Figure 1. Plastic isolator for germ-free husbandry Once in use, an isolator is kept sterile by being ventilated printouts are examined to confirm that the appropriate with HEPA-filtered air under positive pressure. Supplies, cycle was chosen and ran without error. Temperature such as food, bedding, water, and caging, are autoclaved indicators are evaluated and the bioindicators, including in transfer cylinders. To assure sterilization, supplies are those retrieved from inside the test cylinder, are incubated arranged in cylinders following standard configurations (at 55-60 oC). Cylinders are released for use only if all (Figure 2). Self-contained bioindicators containing heat- bioindicators read negative. stable bacterial spores (e.g., EZTest® Steam, Mesa Labs) and temperature indicators are placed throughout each autoclave run, including inside a test cylinder. Autoclave [email protected] • www.criver.com Figure 2. Cylinder for autoclaving supplies 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 3. Autoclaved cylinder attached to isolator Shipping To preserve their germ-free status during shipment, ethylene oxide gas. Upon receipt of a lightweight shipper, mice are transferred from the isolator in which they are the shipper should be attached to the port of the isolator housed through the disinfected double-door lock system and the mice antiseptically transferred. into cages within a germ-free shipper sterilized with C57BL/6NCrl (B6N) Germ-Free Mice Microbiological Monitoring culture media, then incubated aerobically and anaerobically Germ-free colonies are monitored for extraneous bacteria in a dedicated anaerobic workstation. Because microbial and fungi, and for pathogens. Table 2 shows the test contaminants may be fastidious or non-cultivable on methods, samples, and frequencies. Testing for extraneous cell-free media (like much of the indigenous microbiota), microbes is conducted frequently, based on the potentially culture-independent methods are employed. Wet mounts high incidence of this type of contamination
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