Mice
Pet shop mice were purchased from Komodo Reptile. Mice of the following strains were purchased from The Jackson Laboratory: 129S1/SvImJ (strain 002448), A/J (000646), BALB/cJ (000651), CAST/EiJ (000928), C3H/HeJ (000659), C57BL/6J (000664), DBA/1J (000670), FVB/NJ (001800), PERC/EiJ (001307), PWK/PhJ (003715) SJL/J (000686) and WSB/EiJ (001145). C57BL/6J or BALB/cJ mice were used in SPF experiments as indicated. Except where noted, all mice were kept on standard chow diet (Envigo/Inotiv 2018S). In experiments using soy-free diet (Envigo/Inotiv 2020SX), breeder pairs were placed onto soy-free diet and progeny to be used for experiments were weaned onto, and maintained, on a soy-free diet. For experiments using casein diet, breeder pairs and progeny were maintained on a custom 18% casein protein diet (Envigo/Inotiv TD170404). Mice were 8–12 weeks of age at the initiation of experimental procedures except where noted. Experimental cohorts bred in house comprised female and male mice; female mice were used in all other experiments. Cages of mice were randomized across conditions and sample sizes were not determined a priori. Workers were not blinded to treatment conditions. All protocols were reviewed, approved, and conducted under the institutional regulation of Yale University’s Institutional Animal Care and Use Committee.
Pathogen testing and cytokine measurement
Oral swabs, fur swabs and faecal samples were submitted to IDEXX BioAnalytics and screened by PCR for the presence of common mouse pathogens. Serum cytokine measurement was performed by Eve Technologies.
16s rRNA sequencing and analysis
Immediately upon arrival at our facilities, pet shop faecal samples were collected in sterile Eppendorf tubes. DNA was isolated (Qiagen 12855) followed by amplification of the 16S rRNA gene V4 region by PCR using a dual index multiplexing strategy as previously described57. Successful amplification was confirmed by gel electrophoresis; amplicons were then pooled and gel-extracted. Library quantification was conducted on gel-extracted fraction (KAPA Biosystems KK4835; Applied Biosystems QuantStudio 6 Flex instrument). Paired-end sequencing (2 × 250) was performed on an Illumina Miseq using a 500-cycle V2 reagent kit (Illumina MS-102-2003).
Co-housing and fostering
To establish a colony of inbred mice bearing pathogens, adults were co-housed with pet shop mice for two weeks, then separated into breeding cages. Progeny from these breeding pairs were used as experimental animals. For studies on baseline antibody reactivities and T cell memory, adult inbred SPF mice were co-housed with pet shop mice for 60 days and directly tested. For foster experiments, P1 neonatal inbred mice from SPF breeders were fostered onto pet shop dams and remained with the dam until weaning.
Allergic sensitization and anaphylaxis measurement
For subcutaneous (skin) sensitization, 5 µg of low-endotoxin cOVA (Biovendor 321001) adsorbed to ~500 µg (50 µl) aluminium hydroxide gel (alum) (Invivogen vac-alu-250) with PBS to a final volume of 100 µl was injected subcutaneously in the flank near the inguinal lymph node using an insulin syringe (BD 329410). In some experiments, KLH (Sigma H7017) was used as antigen. For intranasal (lung) sensitization, 20 µg of low-endotoxin cOVA and 20 µg of papain (ThermoFisher Scientific 416760100) with PBS to a final volume of 40 µl was administered into the nasal passage of anaesthetized mice. For intragastric (intestinal) sensitization, 50 mg of cOVA grade V (Sigma A5503) and 10 µg of cholera toxin (Sigma 227036) in 200 µl bicarbonate buffer was administered by gavage. Injections or administrations were repeated seven days later, and serum was collected after an additional five days. Two days after serum collection, mice were challenged intraperitoneally with 75 µg cOVA grade V (Sigma A5503) in PBS to a final volume of 200 µl and core body temperature was measured every 15 min using a rectal thermometer (Physitemp TH-5) over the course of 75 min. Perinatal sensitizations were performed with 5 µg cOVA adsorbed to ~250 µg alum in PBS to a final volume of 50 µl injected subcutaneously in the flank.
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