Tips and Tricks for the Care of NSG Mice
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1 Tips and Tricks for the Care of NSG Mice Dominique Kagele, Ph.D. Technical Information Services
2 The Jackson Laboratory s Mission Performing Research Investigating genetics and biology of human disease Providing Resources JAX Mice Clinical & Research Services, bioinformatics data, technical publications and more Educating Scientists World-class courses, internships and other programs 2
3 JAX Mice JAX Mice The The Gold Standard for for Biomedical Biomedical Research Research NIH funded resource >7,500 strains and growing Unsurpassed genetic quality & animal health Best characterized & referenced ~100 new pubs/week Common inbred strains (C57BL/6J, BALB/cJ, DBA/2J) support development/collection of specialty strains and other valuable community research resources 3
4 Online Resources to Expedite Research JAX Mice Database Mouse Genome Informatics Mouse Phenome Database And many more unique resources 4
5 Presentation Overview Handling and Care of the NSG Mouse 5
6 Presentation Overview Learning Goals Explain features and uses of NSG mice Identify the types and signs of common infections in NSG mice Describe the processes and caging recommendations for successful care of NSG mice 6
7 Presentation Overview Learning Goals Explain features and uses of NSG mice Identify the types and signs of common infections in NSG mice Describe the processes and caging recommendations for successful care of NSG mice 7
8 Immune System Components 8
9 A Spectrum of Immune Deficiency Immune deficient Immune competent 9
10 Severely Immune Deficient Strains Fragile Superheroes Nude Rag1/Rag2 scid NOD scid gamma (NSG) 10
11 Immune System Components 11
12 Immune System Components 12
13 Immune System Components 13
14 Immune System Components 14
15 Immune System Components 15
16 NSG, NOD scid gamma: Features Official nomenclature: NOD.Cg-Prkdc scid Il2rg tm1wjl /SzJ (005557) Many Immunological Deficiencies NOD background contributes innate immune deficiencies o o o Macrophages, dendritic cells defective No complement system Bone marrow readily colonized by human hematopoietic stem cells scid mutation prevents development of mature T and B cells Il2rg gene knockout blocks signaling from 6 distinct interleukins and blocks NK cell development Benefits: No scid leakiness Longer lifespan than NOD scid (mean lifespan ~22 months) Highly resistant to thymic lymphoma development, as compared to other scid mutant strains To find NSG information online, visit 16
17 NSG Research Applications An incredibly useful and versatile immunodeficient mouse model Research applications: Primary tumor engraftment Human hematopoiesis Humanized mice Infectious disease Regenerative medicine To find NSG information online, visit 17
18 Presentation Overview Learning Goals Explain features and uses of NSG mice Identify the types and signs of common infections in NSG mice Describe the processes and caging recommendations for successful care of NSG mice 18
19 NSG mice Infectious Disease Concerns High level of immunodeficiency results in extreme susceptibility to: Pathogenic microorganisms: o Infectious agents that typically causes disease in immunocompetent hosts Opportunistic microorganisms: o Potentially infectious agents that rarely cause disease in immunocompetent hosts Commensal microorganisms: o Potentially infectious agents that reside in normal host tissues without causing disease Foreman O, et al Vet Pathol. [PMID: ] 19
20 Pathogenic and Commensal Microbes Common Threats to NSG mice C. bovis Citrobacter Enterobacter Enterococcus spp. Klebsiella spp. Proteus Pneumocystis murina Pseudomonas S. aureus Coagulase-negative Staphylococcus spp. 20
21 Infectious Disease Concerns Opportunistic Infections Opportunistic microorganisms normally present in gut flora of healthy mice can become pathogenic in NSG mice, most commonly: o Klebsiella oxytoca o Enterococcus spp. Clinical Signs: o Hunched posture, scruffy coat o Females (twice as likely to be infected) Foreman O, et al Vet Pathol. [PMID: ] 21
22 Contributing Factors to Mortality Opportunistic Infections Urinary tract infections (UTIs) o Normal intestinal flora o Estrogen supplementation (increased risk) Ascending renal infection and pathology Secondary infection after primary insult o Skin wounds (ie needle punctures, bacterial dermatitis) o Molar gingival sulcus Breeding (often lactating) females more susceptible 22
23 Signs of Infection Commonly Observed in NSG mice Infected skin wounds, cellulitis Abscesses (skin and internal organs) Otitis media, conjunctivitis, panophthalmitis Localized and widespread infections involving liver, heart, lungs, uterus, accessory sex glands, etc. Forman et al Vet Pathol. 48(2): PMID:
24 Infections in NSG mice Contributing Factors Can occur in mice housed under less strict barrier conditions, strict barrier conditions, and isolators Most often seen after mice have been in the facility a while Often appear to be an individual animal vs. colony issue o Sporadic o Sick and healthy (or infected/non-infected) animals commonly in the same cage 24
25 Presentation Overview Learning Goals Explain features and uses of NSG mice Identify the types and signs of common infections in NSG mice Describe husbandry and caging recommendations for successful care of NSG mice 25
26 How Clean is Clean Enough? Decontamination Decontamination Sanitization: Reduction of microbial organisms from inanimate surfaces Disinfection: Destruction or inactivation of most microbial organisms from inanimate surfaces Sterilization: Complete destruction or inactivation of all microbial organisms from inanimate surfaces 26
27 How Clean is Clean Enough? Housing Conditions Barrier practices adequate to maintain nude, or even scid mice may not be adequate for NSG Recommend: o Sanitize hands before gloving (washing) o Disinfect surfaces (laminar flow hoods, experimental equipment, floors, walls) o Sterilize tools (forceps, scissors, ear punches, etc), bedding, and cages o More frequently change cages o Monitor for pathogens frequently (swabbing) 27
28 Care and Handling of NSG mice: Personal Protective Equipment Personal Protective Equipment (PPE) o Sterile scrubs, gloves, dedicated shoes and shoe covers o Mask, goggles, hair/beard bonnet o Sterile smock o PAPR (Powered air purifying respirator): if necessary 28
29 Environmental Conditions: Suggestions Entry room/space (anteroom or dedicated hallway) o Clearly marked with tape and/or signage o Limited entry o Air shower (if available) 29
30 Environmental Conditions Sterilize or disinfect anything that may come into contact with the mice (autoclave or vaporized hydrogen peroxide (VHP)) Use laminar flow hoods (or biosafety cabinet working with human pathogens) whenever possible Micro-isolator/ individually ventilated cages (IVCs) o HEPA (High-efficiency particulate absorption) filtered Cage Changes: o More frequent o Perform in disinfected laminar flow hood o Disinfect gloved hands and anything goes into the hood o Disinfect forceps (Wescodyne) between cages o If hands leave hood, disinfect again 30
31 NSG Mice at JAX: Barrier Conditions Maximum barrier at JAX: Sterilized individually ventilated caging Sterilized feed and drinking water Air shower entry Change into clean room processed scrubs, smock and shoes Gloves, air hat or mask, cap and face shield Under maximum barrier conditions at JAX, bacterial disease in NSG are uncommon less than 1% of mice >200 days Overview of barrier levels at JAX: 31
32 Experimental Procedures and Transport Disinfect experimental equipment and environment (especially shared) Sterilize smaller tools (by autoclaving) MAP test and/or culture material prior to implantation to ensure sterility Use secondary containment during transportation o Plastic bag o Sterile smock 32
33 Breeding NSG mice NOD.Cg-Prkdc scid Il2rg tm1wjl /SzJ (005557) Homozygote/homozygote female x homozygote/hemizygote male No need to genotype pups, but genotype breeders Rotate every 8-9 months Replace if no litters in 60 days or if appear sick 33
34 NSG Mice Care and Husbandry Suggestions Food o Autoclaved and/or irradiated Water o Acidified to ph with HCl (or chlorinated) to control for Pseudomonas spp. o Autoclaved o No antibiotics added routinely Pathogens monitored o Directly (infected mice) o Indirectly (sentinel mice) 34
35 Pathogen monitoring Sentinel mice are those used for the detection of pathogens present in the room, and include: o Dirty-bedding (mostly fecal-oral) o Cage-contact (direct contact, aerosol, urine, fecal-oral) o Exhaust air (aerosolized) Use both immunocompetent and immunodeficient (including NSG periodically) as sentinels For more information, please visit 35
36 Health Monitoring Considerations Expanded health surveillance to detect all organisms excluded from barrier, including opportunistic bacteria o Shedding often intermittent o Non-lethal monitoring, e.g., fecal or oropharyngeal swab culture of colony mice desirable Immune competent mice may be transient carriers of opportunists that cause significant disease in immune deficient (e.g., Corynebacterium, Pneumocystis) o Testing may fail to detect due transient nature o Test immunodeficient mice directly Direct tests (e,g., culture, PCR), not serology, for severely immune deficient 36
37 Immunodeficient Mice at JAX: Animal Health Reports Health Status Reports: 37
38 NSG Mice Treatment of infection Antibiotic treatment of individual mice: o Baytril (enrofloxacin) 5-20 mg/kg SC as directed by your verterinarian o Others (amoxicillin, cephalexin) o No evidence of effectiveness as prophylatic treatment Pneumocystis containment o Sulfa-Trimethoprim: incorporate in feed or water 50 mg/kg/day trimethoprim mg/kg/day sulfamethoxazole o Significant decrease in mortality Fungi may or may not be detectable by histopath, but continue to be detectable by PCR Rederivation to eliminate opportunists from colony Slate AR., et al., JAALAS Marcotte H., et al., J Infect Dis
39 Humanization Health Considerations Possible human pathogens require ABSL2 housing & BSL2 laboratory Possible mouse pathogens from human donor, e.g., LCMV o Test tissues (MAP test, and/or culture bio-materials) Graft vs. host disease severely immune deficient mice attacked by human tissue / cell transplants o Hunched posture, ruffled fur, reduced mobility, tachypnea, diarrhea, weight loss and/or hair loss / skin lesions o Time course (days to weeks) depends on strain of mouse, irradiation preconditioning, and type(s) and numbers of human cells injected 39
40 Warning Signs Non-specific clinical problems Unthriftiness, diarrhea, wasting, sickliness Weight loss Weakness, lethargy Acute and/or premature death Breeding problems, including: o Embryonic death o Small litters o Small, weak, and/or sickly pups o Pup mortality 40
41 Work With Your Veterinarian! When breeding performance suddenly declines Mice appear unhealthy (ruffled fur, hunched, reduced mobility/activity) Spontaneous death Positive culture/sentinel mice results 41
42 Summary A little prevention goes a long way! Handling and Care of the NSG Mouse 42
43 Thank you! In need of mouse breeding and colony management expertise to advance your research? Contact your regional representative today Contact technical support JAX Mice, Clinical & Research Services
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