Marys Medicine



Tips and Tricks for
the Care of NSG

Dominique Kagele, Ph.D.
Technical Information Services

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Handling and
Care of the
NSG Mouse
JAX® Mice
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 JAX® Mice
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 JAX® Mice
Immune System Components
JAX® Mice
A Spectrum of Immune Deficiency
Immune deficient
Immune competent
JAX® Mice
Severely Immune Deficient Strains
Fragile Superheroes
Rag1/Rag2 NOD scid gamma (NSG) JAX® Mice
Immune System Components
JAX® Mice
Immune System Components
JAX® Mice
Immune System Components
JAX® Mice
Immune System Components
JAX® Mice
Immune System Components
JAX® Mice
NSG, NOD scid gamma:

Official nomenclature: NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (005557)
Many Immunological Deficiencies
NOD background contributes innate immune deficiencies
o Macrophages, dendritic cells defective
o No complement system
o 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
No scid leakiness Longer lifespan than NOD scid (mean lifespan 22 months) Highly resistant to thymic lymphoma development, To find NSG information online, visit
as compared to other scid mutant strains JAX® Mice
NSG Research Applications
An incredibly useful and versatile immunodeficient
mouse model

Research applications:
Primary tumor engraftment Human hematopoiesis Infectious disease Regenerative medicine To find NSG information online, visit
JAX® Mice
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 JAX® Mice
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. 2011. Vet Pathol. [PMID: 20817888] JAX® Mice
Pathogenic and Commensal Microbes
Common Threats to NSG mice

Citrobacter Enterobacter Enterococcus spp. Klebsiella spp. Pneumocystis murina Pseudomonas S. aureus Coagulase-negative Staphylococcus spp. JAX® Mice
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. o Hunched posture, scruffy coat o Females (twice as likely to be infected) Foreman O, et al. 2011. Vet Pathol. [PMID: 20817888] JAX® Mice
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 JAX® Mice
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. 2011.Vet Pathol. 48(2):495-9. PMID:20817888 JAX® Mice
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 JAX® Mice
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 JAX® Mice
How Clean is Clean Enough?

inactivation of all inanimate surfaces JAX® Mice
How Clean is Clean Enough?
Housing Conditions

Barrier practices adequate to maintain nude, or even scid mice may not be adequate for NSG o Sanitize hands before gloving (washing) o Disinfect surfaces (laminar flow hoods, experimental equipment, o Sterilize tools (forceps, scissors, ear punches, etc), bedding, and o More frequently change cages o Monitor for pathogens frequently (swabbing) JAX® Mice
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 Environmental Conditions:

Entry room/space (anteroom or dedicated hallway) o Clearly marked with tape and/or signageo Limited entryo Air shower (if available) JAX® Mice
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 JAX® Mice
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: Experimental Procedures and

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 Breeding NSG mice
NOD.Cg-Prkdcscid Il2rgtm1Wjl/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 NSG Mice
Care and Husbandry Suggestions

Food o Autoclaved and/or irradiated Water o Acidified to pH 2.5 - 3.0 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) JAX® Mice
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 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 JAX® Mice
Immunodeficient Mice at JAX:
Animal Health Reports

Health Status Reports: 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 + 250 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. 2014. Marcotte H., et al., J Infect Dis. 1996 JAX® Mice
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 䇾attacked䇿 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 JAX® Mice
Warning Signs
Non-specific clinical problems Unthriftiness, diarrhea, wasting, sickliness 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 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 Summary
A little prevention goes a long way!

Handling and
Care of the
NSG Mouse
JAX® Mice
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THE CHARTERED SOCIETY OF PHYSIOTHERAPY A clinical for the use of injection therapy by This clinical guideline was endorsed by the Chartered Society of Physiotherapy in January 1999. The endorsement process has included review by relevant external experts as well as peer review. The rigour of the appraisal process can assure users of the guideline that the recommendations for

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