| Strain Name |
NOD.CB17-PrkdcscidIl2rgtm1/Bcgen |
Common Name |
B-NDG mice |
| Background |
NOD-scid |
Catalog number | 110586 |
| Aliases |
Male: Prkdc (-/-), Il2rg (X-/Y); Female: Prkdc (-/-), Il2rg (X-/X-) |
||
|
NCBI Gene ID |
16186 | ||
(Click for Chinese Version)
Background
The immunodeficient B-NDG mice (NOD.CB17-PrkdcscidIl2rgtm1/Bcgen) was independently designed and generated by Biocytogen. B-NDG mice were generated by deleting the Il2rg gene from NOD scid mice with severe immunodeficient phenotype. This mouse model lacks mature T cells, B cells and functional NK cells. It is internationally recognized as an immunodeficient mouse model well suited for human-derived tissue or cell engraftment.
- NOD (non-obese diabetes) genetic background: Spontaneous type I diabetes; defective function of T cells, NK cells, macrophages, and dendritic cells and lack hemolytic function of complement.
- Prkdc (protein kinase, DNA activated, catalytic polypeptide) gene mutation: no mature T cells and B cells, severe combined immunodeficiency (scid) of cellular and humoral immunity.
- Il2rg gene knockout: the gamma chain of IL2 receptor (IL2Rγc, CD132) is located on the X chromosome of mouse, and is the common receptor subunit of cytokines IL2, IL4, IL7, IL9, IL15 and IL21 with important immune functions. The immune function of Il2rg knockout mice was severely reduced, especially the activity of NK cells was almost lost.
B-NDG mice: Combined NOD scid-Il2rg null background features, severe immunodeficient phenotype, absence of mature T cells, B cells, and functional NK cells, decreased function of macrophages and dendritic cells. It is very suitable for the transplantation of human hematopoietic stem cells (CD34+ HSCs) and peripheral blood mononuclear cells (PBMC) to obtain humanized mice with human immune system.
• The highest degree of immunodeficiency among all immunodeficient mouse 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
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 new humanized mouse models
Animal breeding and maintenance
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 breeding and maintenance
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)).
Phenotypic analysis
Growth Curve
Three weeks old of newborn pups were obtained at weaning (50 males and 50 females, respectively). Body weight was measured on the same day of every week, and lasted for 12 weeks.
Flow-cytometric Analysis Using Specific Markers for T, B and NK Cells
Blood were collected from BALB/c, NOD scid and B-NDG mice (n=3, 6-week-old, female). Leukocyte subpopulations were analyzed by flow cytometry analysis. A. Representative FACS plots. B. Statistical analysis of FACS. Results showed that T cells, B cells and NK cells were detectable in BALB/c mice. NK cells were detectable in NOD scid mice. But none of the three cells were detectable in B-NDG mice.
Histology of spleen from B-NDG mice
Spleens were isolated from C57BL/6, NOD-scid and B-NDG mice (female, n=3, 9-week-old). The spleen samples were sectioned for HE staining. Results showed that the spleen from C57BL/6 mice has normal structure with well-defined follicles. Spleen from NOD scid mice showed hypoplasia of white pulp. But spleen from B-NDG mice showed complete loss of follicular structure.
Figure 5. Histological sections of spleen and IHC staining.
Spleens were isolated from C57BL/6, NOD scid and B-NDG mice (female, n=3, 9-week-old). The spleen samples were sectioned for IHC staining. Results showed that the spleen from C57BL/6 mice has normal CD3ε and CD19 expression (brown). No CD3ε and CD19 were expressed in NOD scid and B-NDG mice.
Figure 8. Raji B cells (5X106) were injected into each B-NDG, NOD-scid and BALB/C Nude mice.
Drug in vivo efficacy study using Raji lymphoma CDX Tumor metastasis model in B-NDG mice.
Figure 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 disease progression in mice. (B) Tumor curve for tumor cell fluorescence curves in different groups of mice. The effect of early treatment (at day 3, day 10) is remarkable, and this effect is significantly reduced for the late treatment (at day 10).
Human CD47 antibody in vivo efficacy study using Raji lymphoma CDX Tumor model in B-NDG mice.
Human B-luciferase-GFP Raji cells (B lymphocytes) were caudal vein implanted into B-NDG. Mice were grouped when the fluorescence intensity reached approximately 1.0E+06 (n=6) at which time they were treated with anti-human CD47 antibodies with doses and schedules indicated in panel A. (A) fluorescence imager was used to monitor tumor fluorescence in mice. (B) Body weight changes during treatment. The results showed that 3 antibodies had efficacies for tumor growth inhibition in B-NDG mice. B-NDG is a powerful model for human CD47 antibody efficacy study. Values are expressed as mean ± SEM.
PDX Tumor Models Models and Efficacy Evaluation
Note: this is just for presentation use only, we don’t provide PDX directly to the clients.
Blood cancer PDX Models are Successfully Established in B-NDG Mice
Figure 14. Blood cancer PDX models have been successfully established in B-NDG mice.
Flow cytometry of Blood cancer PDX model gated for hCD45 cells and hCD19 positive cells. Result shows that the B-ALL PDX model has been successfully established in B-NDG mice.
Breast Cancer PDX Models are Successfully Established in B-NDG Mice
Figure 15. H&E staining was used to assess tumor/stroma structure of Triple Negative(ER-/PR-/HER2-) Breast PDX samples. Patient-derived xenografts were found to recapitulate the structures in original patient samples and maintain similar heterogeneity in different generations.
Breast Cancer PDX Models are Successfully Established in B-NDG Mice
Figure 16. IHC staining of triple negative(ER-/PR-/HER2-) breast cancer.
IHC staining of PDX samples from triple-negative breast cancer showed that ER, Her2 and PR were negative in the original tumor samples and passage samples, and there was no difference in histomorphological structure between the primary sample and P1-P3 passage samples, fully preserving the heterogeneity of tumor tissues.
Drug in vivo efficacy study using breast PDX tumor model in B-NDG mice.
Figure 17. Drug in vivo efficacy study of kinase inhibitors using triple negative(ER-/PR-/HER2-) breast PDX model.
Drug efficacy on triple-negative breast cancer PDX models via B-NDG mice. (A) The animals were grouped into control and treatment when the tumor size was approximately 150±50 mm3. at which time they were treated with drugs. (B) Body weight changes during treatment. As shown in panel A,Drug X significantly inhibited tumor growth compared with the control group, which proved that the PDX model can effectively evaluate the in vivo efficacy of the anti-cancer drug for triple-negative breast cancer. Values are expressed as mean ± SEM.
PBMC reconstitution model
Figure 18. Analysis of peripheral blood lymphocyte subpopulations by FACS.
Human PBMC cells(5E6) were intravenous implanted into homozygote B-NDG mice(female, 6 week-old, n=6) . Blood from B-NDG mice was taken at different times after implantation of human PBMC for flow cytometric analysis. B-NDG mice showed a high percentage of human CD45+ cells, T cells. B-NDG mice exhibit reduced body weight and shortened survival likely due to GVHD effects caused by a high proportion of human immune cell reconstitution.
Human Immune System Reconstituted Models and Efficacy Evaluation
Figure 19. B-NDG mice reconstituted with PBMCs cells were used for bispecific antibody efficacy studies.
Human B-luciferase-GFP Raji cells (5E5), PBMC (5E6) and antibodies mixture were intravenously injected into B-NDG mice (n=4). (A) fluorescence imager was used to monitor tumor fluorescence in mice. (B) Body weight changes during treatment. Bispecific antibody shows significant inhibitory effects. The results indicate that establishing a CDX tumor model in B-NDG mice with reconstituted PBMCs provide a powerful preclinical model for in vivo evaluation of antibodies. Values are expressed as mean ± SEM.
Human Immune System Reconstituted Models and Efficacy Evaluation
Figure 20. B-NDG mice reconstituted with PBMCs cells were used for CD3×Claudin18.2 bispecific antibody efficacy studies.
NUGC4 cells (5E6) were subcutaneously implanted after human PBMCs (5E6) were intravenous implanted into B-NDG mice (female, 7 week-old, n=6). The animals were grouped into control and treatment when the tumor size was approximately 50-80 mm3 and the percentage of human blood hCD45% were above 10%, at which time they were treated with drugs. (A) Anti human CD3×Claudin18.2 bispecific antibody (AMG 910 analog) inhibited NUGC4 tumor growth in human PBMC reconstituted B-NDG mice. (B) Body weight changes during treatment. CD3×Claudin18.2 bispecific antibody shows significant tumor inhibitory effects. The results indicate that establishing a CDX tumor model in B-NDG mice with reconstituted PBMCs provide a powerful preclinical model for in vivo evaluation of antibodies. Values are expressed as mean ± SEM.
CD34 HSC reconstitution model
Figure 21 . Analysis of peripheral blood lymphocyte subpopulations by FACS.
After 6-week-old mice were irradiated at a dose of 1.2Gy, 1.5×105 CD34+ HSC cells were intravenously injected.. Blood from B-NDG mice was taken at different times after implantation of human CD34+ HSC for flow cytometric analysis. B-NDG mice showed a high percentage of human B cells, T cells, monocytes. Radiator：RS2000pro Biological X-ray Irradiator (Radsource Technologies Inc).
Human Immune System Reconstituted Models and Efficacy Evaluation
Figure 22. B-NDG mice reconstituted with CD34+ cells were used for drug efficacy studies.
Humanized B-NDG mice reconstituted with human CD34+ cells were i.v. injected with Human B-luciferase-GFP Raji cells (5E5).Mice were treated with a human PD-1 antibody 5 days after tumor cell implantation. A dramatic inhibitory effect of the human PD-1 mAb on tumor cell growth was observed at day 7. The results indicate that establishing a CDX tumor model in B-NDG mice with reconstituted HSC provide a powerful preclinical model for in vivo evaluation of antibodies. Values are expressed as mean ± SEM.
NK reconstitution model
Figure 23. Analysis of peripheral blood lymphocyte subpopulations and protein expression on NK cells by FACS. NK cells from normal donors were purified from PBMC via positive selection by magnetic cell-sorting (MACS). The purified NK cells (2×106) were intravenously injected into B-NDG hIL15 mice. Representative result is shown. (A) Blood was collected and analyzed by FACS every week for 7 weeks after NK injection. The absolute numbers and frequencies of human NK cells in mouse peripheral blood are shown. (B) NK related antigen was also detected, which confirmed the reconstituted NK were functional NK. Granzyme: NK cytotoxicity-related protein；NKG2A: NK cell surface inhibitory receptor；NKG2D, NKp46: NK cell surface activated receptors.
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