Immunodeficient B-NDG Mice

Description

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.

  • NOD-scid (non-obese diabetes, severe combined immunodeficiency) genetic background: mice of NOD genetic background and with Prkdc (protein kinase DNA-activated catalytic) knockout. Functional T cells, B cells and the complement system in these mice are lost, and the activity of NK cells is greatly weakened.
  • IL2rg null: the gamma chain of Interleukin-2 receptor (IL-2R γc, also called CD132) is on the mouse X chromosome, and is the common receptor subunit of cytokines IL2, IL-4, IL-7, IL-9, IL-15 and IL-21 with important immune functions. After IL2r is knocked out, mouse immunity function is greatly weakened, activities of NK cells, which are almost completely lost.
  • Prkdc null (DNAPK, scid): Prkdc (protein kinase DNA-activated catalytic) null mutation is characterized by significantly deficient of functional T cells and B cells, and an absence of lymphocytes, recapitulating severe combined immunodeficiency (scid) in human patients.

Advantages of B-NDG Mice

  • Model currently holds the highest degree of immunodeficiency among all immunodeficiency models
  • Longer lifespan than NOD-scid mice; 1.5 years on average
  • Minimal to absent rejection of human-derived cells and tissues
  • More efficient for CDX and PDX model generation
  • No B lymphocyte leakage

Major Applications

  • Human-derived cell or tissue engraftment
  • Tumor and tumor stem cell research
  • ES and iPS cell research
  • Hematopoiesis and immunology studies
  • Human infectious disease studies
  • Development of humanized models

Basic Information

  • Strain name: NOD-PrkdcscidIL2rgtm1/Bcgen
  • Common name: B-NDG mice; B: Biocytogen; N: NOD background; D: DNAPK (Prkdc) null; G: IL2rg knockout
  • Catalog number: B-CM-002
  • Genotypes: Male: Prkdc (-/-), IL2rg (X-/Y); Female: Prkdc (-/-), IL2rg (X-/X-)

Phenotypic Analysis

1. Body Weight Growth

B-NDG-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

B-BDG0Serum-IgG

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 

B-NDG-Serum-IgG-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

B-NDG-Flow-Cytometric

B-NDG-Flow-Cytometric-Bargraph

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

B-NDG-Flow-Cytometric-Analysis

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

B-NDG-Hematology

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

B-NDG-Blood-Biochemical

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
Food

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.

Water

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.

Bedding

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.

Housing Environment

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

2. Transportation

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 1.10.1.2. 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.

CDX Tumor Models

See our CDX Tumor Models here

PDX Tumor Models

See our PDX Tumor Models here

Application for B-NDG Trademark Registration

Beijing Biocytogen Co., Ltd. has applied for the B-NDG trademark registration for its use in different classes of goods/services (Class 5, Class 31, Class 42 and Class 44). Each letter in the name stands for: B-Biocytogen; N-NOD background; D-DNAPK (Prkdc) null; G-IL2rg knockout.

Detailed information of each class (until the date of application):

Class 5: medicines for human purposes; leeches for medical purposes; diagnostic biomarker reagents for medical purposes; preparations of microorganisms for medical or veterinary use; dietetic foods adapted for medical purposes; reagents for veterinary purposes;insecticides;wadding for medical purposes; stem cells for veterinary purposes

Class 31: live animals; poultry, live; fish, live; crustaceans, live; trees; grains [cereals]; plants; seeds for planting / plant seeds; hay; animal foodstuffs

Class 42: quality control; chemical research; biological research; clinical trials; material testing

Class 44: pharmacy advice; hospital services; artificial insemination services; diet and nutrition guidance; animal breeding; veterinary assistance; artificial insemination services (for animals); in vitro fertilization services/in vitro fertilization services (for animals); rental of sanitation facilities; in vitro fertilization services/in vitro fertilization services

B-NDG-Trademark-Registration-1 B-NDG-Trademark-Registration-4 B-NDG-Trademark-Registration-3 B-NDG-Trademark-Registration-2

References

References

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2) Ito M, Hiramatsu H, Kobayashi K, Suzue K, Kawahata M, Hioki K, Ueyama Y, Koyanagi Y, Sugamura K, Tsuji K, Heike T, Nakahata T. 2002. NOD/SCID/gamma(c)(null) mouse: an excellent recipient mouse model for engraftment of human cells. Blood 100(9):3175-82. [PMID: 12384415]

3) Shultz LD, Lyons BL, Burzenski LM, Gott B, Chen X, Chaleff S, Kotb M, Gillies SD, King M, Mangada J, Greiner DL, Handgretinger R. 2005. Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells. J Immunol 174(10):6477-89. [PMID: 15879151]

4) McDermott SP, Eppert K, Lechman ER, Doedens M, Dick JE. 2010. Comparison of human cord blood engraftment between immunocompromised mouse strains. Blood 116(2):193-200. [PMID: 20404133]

5) Lepus CM, Gibson TF, Gerber SA, Kawikova I, Szczepanik M, Hossain J, Ablamunits V, Kirkiles-Smith N, Herold KC, Donis RO, Bothwell AL, Pober JS, Harding MJ. 2010. Comparison of human fetal liver, umbilical cord blood, and adult blood hematopoietic stem cell engraftment in NOD-scid/gammac-/-, Balb/c-Rag1-/-gammac-/-, and C.B-17-scid/bg immunodeficient mice. Blood 70(10):790-802. [PMID: 19524633]

6) Shultz LD1, Brehm MA, Bavari S, Greiner DL. 2011. Humanized mice as a preclinical tool for infectious disease and biomedical research. Ann N Y Acad Sci 1245:50-4. [PMID: 22211979]

7) Covassin L1, Jangalwe S, Jouvet N, Laning J, Burzenski L, Shultz LD, Brehm MA. 2013. Human immune system development and survival of non-obese diabetic (NOD)-scid IL2rγ (null) (NSG) mice engrafted with human thymus and autologous haematopoietic stem cells. Clin Exp Immunol 174(3):372-88. [PMID: 23869841]

8) Wege AK, Schmidt M, Ueberham E, Ponnath M, Ortmann O, Brockhoff G, Lehmann J. 2014. Co-transplantation of human hematopoietic stem cells and human breast cancer cells in NSG mice: a novel approach to generate tumor cell specific human antibodies. MAbs 6(4):968-77. [PMID: 24870377]

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