Animal Models and Cell Lines

>B-NDG Mice

B-NDGTM mice

B:BiocytogenTM;  N:NOD background;  D:DNAPK(Prkdc)null;G:IL2rg knockout.NOD-Prkdcscid IL2rgtm1/Bcgen

B-NDGTM mice are mice with NOD genetic background and Prkdc and IL2rg double gene knockout. They are  internationally accepted mouse model with the highest degree of  immunodeficiency, and they are suitable for human cells or tissue  transplantation.

 1. Basic information about B-NDG mice

1.1.  Strain name

NOD-Prkdcscid IL2rgtm1/Bcgen

1.2.  Common name

B-NDG mice

B: Biocytogen

N: NOD background

D: DNAPK (Prkdc) null

G: IL2rg knockout

1.3.  Catalog number


1.4.  Genotypes

Male: Prkdc (-/-), IL2rg (X-/Y); Female: Prkdc (-/-), IL2rg (X-/X-) 

1.5.  Background

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 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 IL2rg 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. 

1.6. 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

1.7. 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

1.8.    Phenotypic analysis

1.8.1.   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; birthdate +/- 3 days). Body weight was measured once every week (on the same day each week) for 8 weeks.

1.8.2.  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. 

1.9.3.      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 the sera of B-NDG mice, confirming it is an ideal mouse model with severe immunodeficiency.

1.9.4.      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. 

1.9.5.      Flow-cytometric analysis using specific markers for NK cells and macrophages

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 absence of NK cells.

1.9.6.  Hematology test results


Figure 6. Complete blood count test results for B-NDG mice.


Full Name


Full   Name


white blood cell count


neutrophil count


red blood cell count


neutrophil percentage




lymphocyte count


hematocrit or packed cell volume


lymphocyte percentage


mean corpuscle (cell) volume


eosinophil count


mean corpuscular hemoglobin


eosinophil percentage


mean corpuscular hemoglobin concentration


monocyte count


red cell distribution width


monocyte percentage


platelet count


basophil count


mean platelet volume


basophil percentage

 1.9.7.   Biochemical test results for Blood

Instrument:Thermo Fisher scientific # Indiko

Sample:sera or plasma


Figure 7. Blood biochemical test results of B-NDG mice.


Full Name



Alanine transaminase

High level indicates liver injured


Aspartate transaminase

High level indicates liver  damage



High level indicates high blood lipid



High level indicates low glomerular filtration



Hyperglycemia or hypoglycemia  



High level indicates high blood lipid



High level indicates kidney damage, liver disease, diabetes or infection.

1.10.  Animal breeding and maintenance

1.10.1. Animal housing and husbandry 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. 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 sterilizethe 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. Sterilizing 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℃. The temperature difference between day and night should not be more than 4℃.

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.


Range recommended





Ventilating rate

15 times per hr

Light Cycle


Light intensity

15-20 lux (in cage)


≤60 db

1.10.2.      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, the most of the lost body weight is regained (although cannot reach 100%) after 5-7 days of adaptive feeding (Labdiet food is recommended)).

1.10.3.      Adaptive feeding

A. 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.

B. Brief procedure description of adaptive feeding

Perform animal husbandry following 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. 

2.       CDX Tumor Models

2.1. CDX lymph cancer metastasis model in B-NDG mouse

Fig 8. Raji cells (5X106) were injected into B-NDG, NOD-scid and BALB/c Nude mice. We recorded and analyzed the following parameters in the mice at different time points:

(A) Mouse survival after cell inoculation depicted by Kaplan-Merier survival curves.

(B) Body weight (g) change each week after inoculation, calculated weights are relative to weight on the day animals were inoculated.

(C) The percent change of human cells in mouse peripheral blood. Post-inoculation of Raji cells, we took 100 μl of whole blood via retro-orbital venous plexus each week, extracted DNA, and determined the ratio of human cells in peripheral blood of mice by q-PCR.

(D) Compared livers of mice after Raji cell inoculation. Once the weight of the mice was reduced by more than 20% post-inoculation, we euthanized the mice, dissected the viscera, and took pictures.

(E) Immunohistochemical staining of mice viscera after Raji cell inoculation. Once the weight of the mice was reduced by more than 20% post-inoculation, we embedded mouse livers and spleens into wax for immunohistochemical assays. The primary antibody is murine anti-human mitochondrial membrane protein antibody (Millipore, MAB1273).

2.2. Selected established CDX models


2.3.  Drug efficacy study

A                                                                                                                    B


Fig 9. Raji-Fluc cells (5 × 105) were injected into B-NDG mice and the same dose of antibody X was given at day 3 and day 10. (A) In vivo imaging recorded at different time points to observe tumor growth in mice. (B) Tumor curve for tumor cell fluorescence output in different groups of mice. The effect of early treatment (at day 3, day 10) is remarkable; this effect is significantly reduced for the late treatment group (at day 10).


Fig 10. Humanized B-NDG mice reconstituted with human CD34+ cells were i.v. injected with 5x105 Raji-Fluc cells. Mice were treated with human PD-1 antibody 5 days after Raji-Fluc cell implantation. A dramatic inhibitory effect of human PD-1 mAb on tumor cell growth was observed at day 7.

2.4.  Human CD47 antibody efficacy study using B-NDG mice

Figure 11. Efficacy study of human CD47 antibodies in B-NDG mice.

Human B-luciferase-GFP Raji cells (B lymphocytes) were inoculated into B-NDG mice using tail intravenous injection. A fluorescence imager was used to monitor tumor fluorescence in mice. Mice were grouped when the fluorescence intensity reached 1.5E+07 (n=6 for control group and antibody-treated groups, respectively). A) Mean of tumor size±SEM, B) Mean weight±SEM. The results showed that 3 antibodies tested had different efficacies for tumor growth inhibition in B-NDG mice. B-NDG is a powerful model for human CD47 antibody efficacy study. 

3. PDX Tumor Models

In the last 10 years, the wide application of PDX (Patient-Derived Xenograft) has advanced preclinical evaluations and can more accurately predict the clinical efficacy of tumor drugs.

3.1.  B-NDG mice show significant PDX model advantages compared to scid mice


                   A                                                                        B



Gastric Cancer




Tumor size to 250 mm3

Tumor size to 250 mm3

Tumor doubling time



40 days

7 days



56 days

5 days



66 days

10 days











40 days

5 days

Tumor take rate 0%(0/6)

Tumor take rate 66.7%(4/6)

Average doubling time 6.75 days

Fig 12. (A) A patient’s gastric cancer sample was implanted subcutaneously and grown on a B-NDG mouse. (B) Tumor growth curve of the patient’s gastric cancer sample in B-NDG mice. (C) Tumor growth comparison between C.B17 scid and B-NDG mice. 

 3.2. PDX models successfully established on the B-NDG mice (example)

3.3. PDX model based on B-NDG mice is a useful tool to evaluate drug efficacy

Fig 13. Different treatments show varying efficacies on our PDX model from the same patient cancer tissue.

Treatment omparison using large-scale PDX models is very useful to investigate the diverse heterogeneity of human cancers. As a representative of an individual patient, every single PDX model is characterized by distinct genetic and biological properties. Therefore, preclinical trials using multiple PDX models are necessary to evaluate the tumor drug efficacy in different patient populations.

4. Human Immune System Reconstituted Models and Efficacy Evaluation

4.1.  Immune Humanized B-NDG Mice via Human PBMCs Engraftment

Fig 14. Human peripheral blood mononuclear cells (hPBMCs) were caudal vein injected (5x106 cells) into B-NDG mice.The ratio of hCD45+ and mCD45+ was compared after 24 days. hPBMCs amplified well in 3 B-NDG mice and the major subset of hPBMCs reconstitution is T cells. 

 4.2.  Immune Humanized B-NDG Mice via Human CD34+ HSCs Engraftment



Fig 15. Humanization of B-NDG mice with human CD34+ cell engraftment. After irradiation and intravenous injection, the survival rate (A) and body weight (n=5) (B) of the B-NDG mice are shown. Our results suggest that reconstitution via human CD34+ engraftment prolongs the life-span of irradiated B-NDG mice.

Fig 16. High-engraftment efficiency of human CD34+ cells in B-NDG mice. The percentage of human CD45+ cells was measured sequentially at the time points shown after engraftment of 2x105 CD34+ cells into B-NDG mice.

A                                                                                                            B


Fig 17. Representative flow cytometric analysis of PBMCs from mice 10 weeks after engraftment with human CD34+ cells. B-NDG mice show a significantly higher percentage of human CD45+ cells and of multi-lineage cells, including CD3+ T cells. Our results suggest that human B, NK and T cells in reconstituted B-NDG mice were successfully propagated, and there were no humanized cells present in the control group (no injection of CD34+ cells; data not shown).

5.       References

  1. Xinhua Xiao, Huiliang Li, Huizi Jin, Jin Jin, Miao Yu, Chunmin Ma, Yin Tong, Li Zhou, Hu Lei, Hanzhang Xu, Weidong Zhang, Wei Liu, and Yingli Wu. 2017. Identification of 11(13)-dehydroivaxillin as a potent therapeutic agent against non-Hodgkin's lymphoma. Cell death & disease. 8(9):e3050. 

  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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