The Immune-deficient B-NDG mouse model (NOD-Prkdcscid IL2rgtm1/Bcgen) was independently designed and generated by Biocytogen. B-NDG mice are generated by deleting the IL2rg gene from NOD-scid mice with severe immunodeficiency phenotype. Lacking mature T cells, B cells or functional NK cells, and displaying cytokine signaling deficiencies, this mouse model has the highest degree of immunodeficiency and thus is most suitable for engraft and growth of human hematopoietic stem cells (HSCs), peripheral blood mononuclear cells (PBMCs) and human tumor cells or tissues.
1. Body Weight Growth
Figure 1. Body weight growth curve of B-NDG mice after birth
Newborn pups (50 males and 50 females, respectively) were obtained at weaning (Week 3; birth date +/- 3 days). Body weight was measured once every week (on the same day each week) for 8 weeks.
2. Serum Antibody (IgG and IgM) Response
Figure 2. IgG and IgM response in the sera of BALB/c, B-NDG and PBS
Value of OD450 in the sample from BALB/c mice is significantly higher than that from PBS and B-NDG mice (~0.04), indicating little or no IgG or IgM in the sera of B-NDG mice.
3. Serum Antibody (IgG subclasses) Response
Figure 3. IgG subclasses response in the sera of BALB/c, B-NDG and 1%BSA
Value of OD450 in the sample from BSA is ~ 0.06 (baseline is 0.1), indicating there is no cross-reaction among antibody capture, linking of enzyme to the antibody and BSA. Compared with the value from BALB/c mice, the value from B-NDG mice is below the baseline. This result suggests that there is no IgG subclasses in sera of B-NDG mice, confirming it is an ideal mouse model with severe immunodeficiency.
4. Flow-cytometric Analysis Using Specific Markers for T, B and NK Cells
Figure 4. Loss of T, B and NK cells in B-NDG mice.
(A) Splenocytes of BALB/c, NOD-scid and B-NDG mice were isolated. Fractions of T, B and NK cells were characterized using flow-cytometry. (B) Statistical analysis of sorted cells.
5. Flow-cytometric Analysis Using Specific Markers for NK Cells
Figure 5. NKp46 expression in splenocytes and blood cells.
NKp46 expression was detected in splenocytes and blood cells of C57BL/6 mice, but not in B-NDG, indicating the absence of NK cells.
6. Hematology Test Results
Figure 6. Complete blood count test results for B-NDG mice.
Abbreviation Full Name Abbreviation Full Name WBC white blood cell count NE# neutrophil count RBC red blood cell count NE% neutrophil percentage Hb hemoglobin LY# lymphocyte count HCT hematocrit or packed cell volume LY% lymphocyte percentage MCV mean corpuscle (cell) volume EO# eosinophil count MCHC mean corpuscular hemoglobin EO% eosinophil percentage MCH mean corpuscular hemoglobin concentration MO# monocyte count RDW red cell distribution width MO% monocyte percentage PLT platelet count BA# basophil count MPV mean platelet volume BA% basophil percentage
7. Biochemical Test Results for Blood
Instrument: Thermo Fisher scientific # Indiko
Sample: sera or plasma
Abbreviation Full Name Explanation ALT Alanine transaminase High level indicates liver injured AST Aspartate transaminase High level indicates liver damage CHOL Cholesterol High level indicates high blood lipid CR Creatinine High level indicates low glomerular filtration GLU Glucose Hyperglycemia or hypoglycemia TRIG Triacylglycerol High level indicates high blood lipid UREA Urea High level indicates kidney damage, liver disease, diabetes or infection.
Animal Breeding and Maintenance
1. Animal Housing and Husbandry
1.1 Health Status of Housing
Health status of housing: B-NDG mice are housed in isolators instead of IVCs in our facility. Based on our experience, the mice can live up to 2 months in SPF standard IVCs. This time frame matches the requirements of most experiments performed with B-NDG mice. To improve facility standards, strict sanitation procedures are recommended: cages and bedding need to be sterilized by autoclaving or Co60 irradiation before use, and cages need to be changed in laminar flow hoods weekly. Keeping a clean, high standard housing environment helps to improve the life span of B-NDG mice.
1.2. Animal Husbandry
5CJL from Labdiet (USA) is recommended to use for breeding B-NDG mice (19.3% protein, 6.2% fat, 20 ppm Vitamin K). Co60 radiation is recommended to sterilize the food before use.
B-NDG mice are housed in pathogen-free isolators in our facility. Autoclaved purified water is used.
For SPF standard facilities, we recommend following the Jackson Lab standard for water supply: acidified water (adjust pH to 2.5-3.0 using HCl), autoclaved to prevent Pseudomonas and Staphylococcus aureus infection. Autoclaved purified water can also be used with more frequent water changes. Bottle must be changed every 3 days regardless if there is still water left in the bottle.
Shavings are the recommended bedding material for B-NDG mice. The bedding material needs to be sterile, soft, dust-free, odor-free and have high moisture absorbance. Sterilization by autoclaving or irradiation is required before use.
Bedding needs to be changed weekly in laminar flow hoods if the mice are not housed in isolators. Mice need to be transferred into new cages with fresh bedding using sterile tweezers or forceps.
Enough light time and appropriate light intensity are necessary for breeding. We use a standard light cycle, which is 12-hours of light followed by 12-hours of dark.
Housing temperature is strictly 20-26 °C. The temperature difference between day and night should not be more than 4 °C.
Cages need to be made from non-toxic material and must be easy to clean and disinfect. Thorough cleaning and disinfection is required every week at least.
Parameters Range recommended Temperature 20℃-26℃ Humidity 40%-70% Ventilating rate 15 times per hr Light Cycle 12:12（standard） Light intensity 15-20 lux (in cage) Noise ≤60 db
Biocytogen’s B-NDG mouse can be shipped using land and/or air. Although the courier is notified to handle the crate with care, stress response of mice during shipment is still inevitable. Although enough supply of water jelly and food will be provided in cages, increased metabolism and fecal excretion caused by the stress may result in dehydration and loss of body weight. General percentage weight loss due to shipment is ~10%. The percentage can be as high as 15% if the shipment procedure is longer and the cage is populated. Usually, most of the lost body weight is regained (although cannot reach 100%) after 5-7 days of adaptive feeding (Labdiet food is recommended).
3. Adaptive Feeding
Importance of adaptive feeding
Before performing experiments, at least 5-7 days of feeding in the receiving facility are required so that the animals can adapt to their new environment, and the stress response caused by transportation can be eliminated or alleviated.
Brief procedure description of adaptive feeding
Perform animal husbandry following 184.108.40.206. Monitor the health status of animals by observing their appearance (e.g. hair), feces and activity. Separate the animals from other animals in the facility as the sound and smell (e.g. Ammonia smelling feces) from other animals may be stimuli. Adaptive feeding is a critical prerequisite for successful experiments.
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