NKG

1. Detection of Prkdc gene mutation

Figure 1. Mutation type detection of Prkdc gene in NKG mice. The Prkdc^scid mutation was generated by TAT→TAA transition in exon 84 of the Prkdc gene, and gene sequencing results showed that NKG mice carried the Prkdc^scid mutation.

2. Detection of Il2rg knockout

Figure 2. Deletion of Il2rg gene in NKG mice. PCR assay of the Il2rg gene in NKG mice showed successful deletion of the Il2rg gene in NKG mice (Band size of wild-type Il12rg gene: 1430bp; band size of Il12rg gene with partial fragment knocked out: 388bp).

3. Detection of B, T, and NK cells in the Peripheral Blood (PB) of NKG mice

Figure 3. NKG mice showed severe immunodeficiency with severe deficiency of B, T, and NK cells in peripheral blood (PB). The results showed that B cells (CD3-CD19+), T cells (CD3+CD19-), T helper cells (CD3+CD4+CD8-), cytotoxic T cells (CD3+CD4-CD8+), and NK cells (CD335+CD3-) were almost completely absent in the peripheral blood of NKG mice.

4. Detection of B, T, and NK cells in the spleen of NKG mice

Figure 4. NKG mice showed severe immunodeficiency with severe deficiency of B, T, and NK cells in the spleen. The results showed that B cells (CD3-CD19+), T cells (CD3+CD19-), T helper cells (CD3+CD4+CD8-), cytotoxic T cells (CD3+CD4-CD8+), and NK cells (CD335+CD3-) were almost completely absent in the spleen of NKG mice.

5. NKG mice for the study in human PBMC (huPBMC) humanized immune system

a. Survival curve

Figure 5. The survival curve of huPBMC-NKG mice. Around 35 days after transplantation, huPBMC-NKG mice gradually began to die as graft-versus-host disease (GvHD) appeared. After 60 days of transplantation, about 20% of the mice were still alive.

b. Proportion of human leukocytes

Figure 6. The proportion of human CD45+ cells in peripheral blood (PB) of huPBMC-NKG mice. After transplantation of human PBMCs, the content of human leukocytes in the peripheral blood of NKG mice gradually increased. After 3 weeks, the average proportion of human CD45+ cells exceeded 40%, and the reconstruction speed was relatively fast (similar products generally take 4 weeks to reach the same level of humanization ratio). After transplantation, the humanization ratio remained at a high level for 3-6 weeks.

c. Proportion of T-cell reconstitution

Figure 7. The proportion of human CD3+ T cells in the peripheral blood of huPBMC-NKG mice. About 95%- 100% of hCD45+ cells in NKG mice belong to hCD3+ T cells 3 weeks after human PBMC transplantation, indicating that the reconstruction of the human immune system in huPBMC-NKG mice was dominated by T cells.

d. The Graft-versus-Host Disease (GvHD)

Figure 8. GvHD scores and body weight changes in NKG mice after PBMC transplantation. GvHD symptoms begin to appear around 28d. Mice lost a significant amount of body weight, and the body weight was reduced by more than 20% after 45d.

6. NKG mice (Adult) for the study in huCD34+ HSC (huHSC) humanized immune system

a. Survival curve

Figure 9. The survival curve of huHSC-NKG adult mice. The huHSC-NKG adult mice were free of graft-versus-host disease (GvHD) and maintained a high survival rate of over 70% after 150 days post-engraftment.

b. Growth curve

Figure 10. The growth curve of huHSC-NKG adult mice. The huHSC-NKG adult mice maintained normal weight gain after the transplantation of huHSC.

c. Proportion of human leukocytes

Figure 11. The proportion of human CD45+ cells in peripheral blood of huHSC-NKG adult mice. The percentage of human leukocytes gradually increased, and the average percentage of human CD45+ cells reached more than 60%.

d. Proportions of human T cells and B cells

Figure 12. Proportions of human T cells and B cells in huHSC-NKG adult mice. The reconstruction of the immune system in this model was dominated by B cells in the early stage. The proportion of B cells gradually decreased and the proportion of T cells gradually increased in the later stage. At 12 weeks post-engraftment, the proportion of T cells began to increase, reaching about 40% at week 16. The proportion of B cells was higher at the early stage, reaching 80%, and decreased to 15% at week 24.

e. Proportions of human NK cells and monocytes

Figure 13. Proportions of human NK cells and monocytes in huHSC-NKG adult mice. The proportions of NK cells and monocytes during immune system reconstitution were low. The average proportion of NK cells maintained at about 2% overall and the proportion of monocytes at week 16 was about 2%.

7. NKG mice (Newborn) for the study in huHSC humanized immune system

a. Survival curve

Figure 14. The survival curve of huHSC-NKG newborn mice. The huHSC-NKG newborn mice were free of graft-versus-host disease (GvHD). The survival rate of huHSC-NKG newborn mice maintained more than 90% before 100 days post engraftment and the survival rate still maintained more than 80% nearly 150 days post engraftment, which was higher than that of the adult model.

b. Growth curve

Figure 15. The growth curve of huHSC-NKG newborn mice. The huHSC-NKG newborn mice maintained normal weight gain after the transplantation of huHSC.

c. Proportion of human leukocytes

Figure 16. The proportion of human CD45+ cells in peripheral blood of huHSC-NKG newborn mice. The percentage of human leukocytes gradually increased. The average percentage of human CD45+ leukocytes reached more than 40% after 8 weeks post engraftment and subsequently increased and maintained at about 60%.

d. Proportions of human T cells and B cells

Figure 17. Proportions of human T cells and B cells in huHSC-NKG newborn mice. Similar to the adult model, the reconstruction of the immune system in this model was dominated by B cells in the early stage. The proportion of B cells gradually decreased and the proportion of T cells gradually increased in the later stage. At 12 weeks post-engraftment, the proportion of T cells began to increase, reaching about 40% at week 14. The proportion of B cells was higher than 80% at the early stage and decreased continuously, dropping to 40% at week 20.

e. Proportions of human NK cells and monocytes

Figure 18. Proportions of human NK cells and monocytes in huHSC-NKG newborn mice. The proportions of NK cells and monocytes were higher in this newborn model than that in the adult model. The average percentage of NK cells was more than 10% in the early stage and maintained at about 5% in the later stage. The average percentage of monocytes was higher than 5%. Therefore, the newborn model is more suitable for studies related to myeloid cell targets.

8. NKG mice for the establishment of cell line-derived xenograft (CDX) models

Figure 19. Tumor growth curves after subcutaneous xenografts of various human-derived tumor cells.Tumor cells were inoculated into NKG and NOD-Scid mice by subcutaneous injection at a cell inoculation rate of 5x10^6/each, and tumorigenic volumes were measured at different time points. The results showed that human pancreatic cancer cell line PANC-1, human liver cancer cell line Huh7, human gastric cancer cell line HGC-27, and human colon adenocarcinoma cell line SW620 were effective in establishing tumor models on NKG mice. The rate of tumor growth, as well as size in NKG mice, were better than those in NOD-Scid mice.

9. NKG mice for the establishment of a hematological tumor cell transplantation model

Figure 20. Tumor growth in NKG mice after transplantation of human Jurkat cells. Transplant human T-lymphocyte cell line Jurkat with luciferase marker into NKG mice by tail vein injection and subsequently detect tumor growth by fluorescence generation. The results showed that Jurkat lymphocyte cells could grow in NKG mice, indicating the successful establishment of this tumor model.

10. The anti-tumor pharmacodynamics of paclitaxel on the human lymphoma (Raji-Luc) CDX model

Figure 21. The anti-tumor pharmacodynamics of paclitaxel on the human lymphoma (Raji-Luc) CDX model. Transplant human lymphoma (Raji-Luc) cell lines into NKG mice and treat with the anti-cancer drug paclitaxel (PTX) to verify the effect of tumorigenesis in NKG mice and the pharmacodynamic. The results showed that the human lymphoma (Raji-Luc) cell line could grow normally in mice and lead to their death by day 28. In contrast, treatment with paclitaxel (PTX) effectively attenuated tumor development and maintained the survival of mice.

11. Establishment of PANC-1 pancreatic in-situ model using NKG mice

Figure 22. Establishment of a pancreatic in-situ model using NKG mice. After transplanting the human pancreatic cancer cell line PANC-1 into NKG mice, the fluorescent effect, mouse survival, and average total fluorescence value were measured. The results show that the PANC-1 pancreatic in-situ model was successfully established.

12. Establishment of NCI-H441 lung in-situ model using NKG mice

Figure 23. Establishment of a lung in-situ model using NKG mice. After transplanting the human lung cancer cell line NCI-H441 into NKG mice, the fluorescent effect and the average total fluorescence value were measured, and the lung tissue of the mice was observed by dissection. The results show that the NCI-H441 lung in-situ model was successfully established.

13. Establishment of MCF-7-luc breast cancer bone metastasis mouse model using NKG mice

Figure 24. Establishment of a breast cancer bone metastasis mouse model using NKG mice. Human breast cancer MCF-7-luc cells were inoculated into NKG (left) in the form of tibial injection, with a cell inoculation amount of 0.5×106 per mouse. At the same time, MCF-7-luc cells were inoculated into NKG in the form of iliac artery injection, with a cell inoculation amount of 1×106 per mouse (right). The results show that MCF-7-luc can effectively establish a bone metastasis tumor model in NKG mice.

14. H&E Staining

a. H&E staining of various tissues of NKG mice

Figure 25. Pathological examination of NKG mice. No obvious abnormalities were observed in the lung, liver, heart, kidney, stomach, small intestine, colon, and cervix. However, the spleen lacked white pulp, and lymphocytes were reduced in the spleen and thymus.

b. H&E staining results of spleen and thymus tissues

Figure 26. Pathological examination of spleen and thymus tissues in wild-type (WT) mice, NOD-Scid mice, and NKG mice. Results show that the spleen structure of wild-type mice is normal, with intact follicles and a normal red-to-white pulp ratio. In contrast, NOD-Scid and NKG mice exhibit a decreased number of lymphocytes and unclear red and white pulp boundaries in the spleen. Additionally, the thymus structure of wild-type mice is normal, whereas the thymuses of NOD-Scid and NKG mice are atrophied, with significantly reduced cortical and medullary lymphocytes.

15. Complete Blood Count (CBC) of 8-week-old NKG mice (n=20, data presented as Mean±SEM)

16. Blood biochemistry (n=8, data presented as Mean±SEM)

17. In vivo tumorigenic validation of human-derived tumor cell lines

*NOD-Scid mice (catalog number: C001070) are immunodeficient mice constructed by introducing the Prkdc^scid mutation on a non-obese diabetic mouse (NOD) background strain with innate T and B lymphocyte immunodeficiency. BALB/c Nude mice (catalog number: C001217) are immunodeficient mice lacking mature T lymphocytes.

*The specific validation data in this table can be found on the official website.