Frequently Asked NSG™ Questions
Below are common questions and answers for maintaining and using NSG™ mice in biomedical research. The questions are organized under the following sections:
- Basic facts about NSG™ mice
- Comparisons with other strains
- Husbandry and handling
- Humanized NSG™ mice
Basic facts about NSG™ mice
How immunodeficient are NSG™ mice?
NSG™ is one of the most immunodeficient mouse strains described to date. Here's why:
- The NOD genetic background contains alleles that reduce the function of the innate branch of the immune system. Consequently, macrophages and dendritic cells are defective.
- scid is a loss-of-function mutation of the Prkdc gene that prevents the development of T and B cells. Prkdc encodes the catalytic subunit of a DNA dependent protein kinase with a role in resolving the DNA double strand breaks that occur during V(D)J recombination. In the absence of V(D)J recombination, the T cell receptor (TCR) gene in T cells and the immunoglobulin (Ig) gene in B cells are not expressed, and T and B cells cannot mature.
- The gamma chain of the interleukin 2 receptor (Il2rg) is a common component of the cell surface receptors for six different interleukins (IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21). NSG™ mice have a complete null mutation (knockout) of this gene. The signaling pathways for these cytokines are blocked in Il2rg knockout mice, although the cytokines themselves are still present. The major consequence of Il2rg deficiency is an absence of functional NK cells, which require IL15 signaling to develop.
How long do NSG™ mice live?
NSG™ mice can live over 1.5 years in a sufficiently clean environment (Prof. Lenny Shultz’s first publication about the strain reports a median survival time that exceeds 89 weeks). They are susceptible to opportunistic pathogens, as discussed below. They live longer than other scid mice because they do not develop thymic lymphoma (the major cause of death of the parental strain, NOD scid).
Which immune cells remain in NSG™ mice?
Neutrophils and monocytes constitute most of the remaining mouse immune cells detectable in peripheral blood. Dendritic cells and macrophages are also present in the mouse, although they are defective because of alleles in the NOD/ShiLt genetic background.
What does it mean for NSG™ mice to be “radiation sensitive”?
The gene Prkdc, mutated by scid, enocodes a DNA protein kinase that participates in DNA double strain break repair throughout the body, and not just in developing immune cells. Consequently, mice that carry the Prkdcscid mutation have increased sensitivity to chemical or physical agents that damage DNA, such as cancer chemotherapies and irradiation. Mice expressing scid require a lower dose of preconditioning irradiation, compared with mice harboring the Rag1 knockout. On the other hand, they do not tolerate very high doses of radiation. NSG™ mice tolerate radiation doses up to 400 cGy (4 Gy). The radiation sensitivity of NSG™ mice might become an issue when studying the response of an engrafted tumor to high-dose radiation treatment. Furthermore, chemotherapies that act by causing DNA damage can have higher toxicity in scid mice, compared to Rag1 or Rag2 knockouts. A maximum tolerated dose study is advisable before dosing NSG™ mice with any chemotherapy.
Are NSG™ mice susceptible to streptozotocin (STZ)?
Yes, NSG™ mice are susceptible to STZ. STZ is an alkylating agent that kills the insulin producing beta cells in the pancreas, resulting in a state that resembles the end stage of type 1 diabetes.
Where can I go to find more information about NSG™?
- The JAX strain datasheet has basic information including genotyping protocols.
- “Breakthrough Research Using NSG™ Mice” lists an annotated, categorized list of publications that is updated weekly.
- Review the original publication describing the strain.
- Contact Technical Information Services to discuss further how NSG™ might further your specific research goals
Comparisons with other strains
What is a “scid-beige” mouse, and how similar is it to NSG™?
A “scid beige” mouse expresses the same scid mutation found in NSG™, along with the “beige” mutation that impairs NK cells by reducing their degranulation capabilities. The genetic background is congenic with BALB/c. The level of immunodeficiency of a scid-beige is probably similar to NOD-scid, but not as high as NSG™. NSG™ is a better host for humanized immune systems than scid-beige. scid beige mice are not available from The Jackson Laboratory.
How does NSG™ compare to other strains used in the field?
Most direct comparisons pertain to “humanized mice”. NSG™ is superior over other models for human CD34+ (stem cell) and PBMC (mature immune cell) engraftment. Inferior models include:
- Any strain expressing the scid mutation alone (NOD-scid, B6-scid, C.B17-scid)
- “scid-beige”
- The same mutations—scid (or a Rag1 or Rag2 knockout) and Il2rg - on other backgrounds (for example, BALB/c)
- References:
How does scid differ from knockouts of Rag1 or Rag2?
Mice with a knockout of either Rag1 or Rag2 have a very similar phenotype in the immune system (elimination of T and B cells), but they do not have the side effect of radiation/chemotherapy sensitivity. Rag1 and Rag2 knockout mice have essentially the same phenotype, and a knockout of either gene suffices to eliminate the adaptive immune system.
How does NSG™ compare to NRG? Why should I use one over the other?
NSG™ and NRG (NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ, 007799) are very similar strains. NRG mice substitute the Rag1 knockout mutation for the scid mutation. The Rag1 knockout has a very similar phenotype in the immune system (elimination of T and B cells), but it does not have the scid side effect of radiation/chemotherapy sensitivity.
There is one publication comparing NSG™ to NRG in a humanized immune system model created by injection of human CD34+ stem cells. The recovery of mature human immune cells is essentially the same.
NRG mice do not have the same sensitivity to DNA damage that NSG™ mice do. NRG could be used in any application that requires especially high doses of radiation. Note that NSG™ mice do tolerate the radiation doses necessary for human hematopoietic stem cell engraftment. NSG™ mice do not tolerate radiation doses at or above 400cGy (4 Gy), while NRG mice tolerate radiation at doses up to 650 cGy. Many chemotherapies act by damaging DNA, and the scid mutation also makes a mouse more sensitive to the side effects of chemotherapies. Although In Vivo Pharmacology Services has successfully dosed NSG™ mice with many different chemotherapies (cisplatin, carboplatin, araC, and others), there may be some instances when NRG mice are preferred if the treatment remains persistently toxic to the mouse.
Husbandry and handling
What pathogens are NSG™ mice susceptible to, and how do they get infected?
NSG™ mice are severely immunodeficient and unable to fight off infections. They are susceptible to infections by normal mouse pathogens, opportunistic pathogens, and even their own intestinal flora. They can become infected from injection sites, bite wounds, and any insult that punctures the skin. The most common type of infection is an ascending urinary tract infection.
How do I keep NSG™ mice healthy?
Proper aseptic handling techniques are essential when working with NSG™ mice. For more details, please see the “Housing and breeding considerations for NSG™ mice” the FAQ section on this page.
Do NSG™ mice breed well?
Yes, assuming that they are maintained in a sufficiently clean environment. Poor breeding performance can indicate an infection. For more details, please see the “Housing and breeding considerations for NSG™ mice” the FAQ section on this page.
Do NSG™ mice need antibiotics in their food or water?
We do not maintain NSG™ mice on antibiotics at The Jackson Laboratory. We find that strict adherence to proper husbandry and handling practices can prevent the introduction of pathogens to the mice, and ensure their long-term survival.
Humanized NSG™ mice
Why are NSG™ mice the best hosts for human hematopoietic stem cells?
- Lack of immune cells. NSG™ mice are missing T, B, and NK cells. NK cells are a major barrier against the efficient engraftment of human hematopoietic stem cells.
- They have a Sirpa polymorphism. Sirpa encodes a protein called Sirpα that is expressed on the surface of macrophages in the bone marrow. Human hematopoietic stem cells express the CD47 surface marker, which binds to Sirpα during engraftment in the bone marrow. High affinity binding of the Sirpα to human CD47 inhibits the mouse macrophages from engulfing the human hematopoietic stem cells. All strains with the NOD genetic background express a variant of Sirpα that has high affinity for human CD47, which results in better engraftment.
- The scid mutation destabilizes mouse hematopoietic stem cells. This is thought to improve the engraftment of human hematopoietic stem cells in the bone marrow stem cell niche.
What is a “humanized mouse”?
A “humanized mouse” refers either to a mouse that expresses a human gene, or one that contains human cells or tissues. The most common type of humanized NSG™ mouse is one carrying human immune cells that have either been generated in the mouse (following CD34+human hematopoietic stem cell injections), or generated in a human donor and injected into the mouse (PBMCs).
What are CD34+cells and PBMCs?
CD34 is a marker for stem/progenitor cells that are capable of producing every hematopoietic lineage. When injected in a NSG™ mouse, they naturally migrate to the bone marrow and differentiate into the mature cell types of the immune system, along the established progenitor pathways.
PBMCs (“peripheral blood mononuclear cells”) include mature lymphocytes (B, T, NK cells), monocytes and macrophages. When injected in the NSG™ mouse, PBMCs either remain in circulation (T cells), or die/migrate to other tissues (all other cell types). They are collected from a blood donation, usually from healthy donors, but can be from diseased or sick patients.
Why do NSG™ mice require irradiation before hematopoietic stem cell engraftment?
Treatment with radiation (usually from an X-irradiator or a cesium source) is a prerequisite for efficient colonization of mouse bone marrow by human hematopoietic stem cells. Irradiation works by killing the mouse stem cells and opening the bone marrow niche, and also by inducing expression of cytokines like Kit ligand (also known as stem cell factor, or SCF). The preconditioning irradiation dose depends on the age of the mouse and often needs to be optimized in every laboratory. Newborn mice tolerate lower doses than juvenile or adult mice.
Are humanized mice available from The Jackson Laboratory?
Yes, through In Vivo Pharmacology Services.
How long can a CD34-humanized NSG™ mouse remain engrafted with human immune cells?
CD45+ cells (mature white blood cells) have been detected in the peripheral blood as long as one year after injection with CD34+ hematopoietic stem cells. In the experience of JAX In Vivo Pharmacology Services, there are no signs of graft-versus-host disease when T-cell-depleted stem cells are sourced from cord blood in mice for up to one year post-engraftment.
How functional is the human immune system that develops in a CD34-injected NSG™?
The different cell types that make up the lymphoid and myeloid lineages are present within humanized NSG™ mice, and there is a significant amount of effort going into understanding how functional they are. Here’s a summary of some key findings:
- The T cell repertoire is diverse and can participate in delayed type hypersensitivity responses.
- B cells are present and can show some immune responses, although they are not especially robust because of the absence of human cytokines that support this lineage.
- The functions of the NK cells, neutrophils, red blood cells, and other cells of the myeloid lineages have all been published.
- JAX In Vivo Pharmacology Services has also observed a cytokine burst characteristic of Th1 and Th2 responses following LPS administration.
- The humanized immune system is capable of rejecting HLA-mismatched human pancreatic islet transplants as well as mouse skin grafts.
Other analyses of the different lineages can be found in the online categorized list of references.
What is graft-versus-host disease? When does it occur?
Graft-versus-host-disease (GVHD) occurs when mature immune cells mount an immune attack on the mouse. This is a possibility any time the immune cells are sourced directly from human blood (PBMCs). It also happens when mature mouse immune cells are injected, if the cells come from any strain with a a major histocompatibility complex (MHC) haplotye that is not matched to NOD/ShiLt. GVHD usually sets in within 3-4 weeks (depending on cell dose and whether or not mice were irradiated) after injection of human PBMCs. NSG™ mice without MHC class I show delayed onset of GVHD.
When can a humanized NSG™ mouse be used for vaccine studies?
To function in a vaccine model, the human T cells in the mouse must be able to interact efficiently with human antigen presenting cells, such as dendritic cells. This phenomenon is known as “HLA restriction” (HLA is the human counterpart to the mouse MHC). Unless the human T cells have developed in a transgenic mouse expressing human HLA, or in a mouse with a human thymus implant, then the interactions are not efficient, and the humanized immune system is probably not capable of mounting an efficient immune response to a vaccination.
What are HLA (human MHC) transgenic NSG™ mice? What are their research applications?
Expression of human MHC (“HLA”) class I improves the function of cytotoxic T cells (CD8+ cells). This is useful for studies involving infectious diseases that infect human immune cells (Epstein-Barr virus, for example), because this response is largely controled by cytotoxic T cells. NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(HLA-A2.1)1Enge/SzJ (Stock # 009617) and NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(HLA-A/H2-D/B2M)1Dvs/SzJ (Stock # 014570) are two strains that express the HLA-A2.1 class I haplotype.
Expression of human MHC (“HLA”) class II improves the function of helper T cells (CD4+ cells). This should be useful for vaccine studies. NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(HLA-DRA*0101,HLA-DRB1*0101)1Dmz/GckRolyJ (Stock # 012479) and NOD.Cg-Prkdcscid Il2rgtm1Wjl H2-Ab1tm1Gru Tg(HLA-DRB1)31Dmz/SzJ (Stock # 017637) are two examples. The second strain does not express the endogenous mouse MHC class2 complex.
Do NSG™ mice require irradiation before injecting PBMCs?
No. Only hematopoietic stem cells require preconditioning irradiation for efficient engraftment. Irradiation accelerates the GVHD response.
Where can I find protocols describing humanized NSG™ mice?
Below is a list of publications that describe protocols and considerations for creating humanized NSG™ mice. Pearson, et al. 2008 is especially recommended.
- McDermott SP, et al. 2010. Comparison of human cord blood engraftment between immunocompromised mouse strains. Blood. Jul 15;116(2):193-200 [PubMed ID: 20404133]
- Brehm MA, et al.. 2010. Parameters for establishing humanized mouse models to study human immunity: analysis of human hematopoietic stem cell engraftment in three immunodeficient strains of mice bearing the IL2rgamma(null) mutation. Clin Immunol. Apr;135(1):84-98. [PubMed ID: 20096637]
- Pearson T, et al. 2008. Creation of "humanized" mice to study human immunity. Curr Protoc Immunol. May; Chapter 15:Unit 15.21. [PubMed: 18491294]
Applications in Cancer Research
Which cancer xenograft models benefit from using NSG™ mice?
Certain cancer models are established more efficiently in NSG™ mice compared to other strains:
- Hematopoietic cancers (leukemia in particular) engraft significantly better in NSG™ compared to other strains. That includes leukemia cell lines and patient-derived samples.
- Most solid cancer cell lines probably do not require the severe immunodeficiency of NSG™; however, you may find that cell lines that do not grow well in other strains grow better in NSG™.
- ER+ breast cancer xenografts, for reasons discussed below.
Can NSG™ mice support the growth of patient-derived or clinical tumor samples?
Yes. JAX In Vivo Pharmacology Services has had great success establishing diverse types of patient-derived tumor models in NSG™ mice, including leukemia models. Slow-growing tumors benefit from the long lifespan of NSG™ mice. Lungand ovarianPDX models are described in the literature, as is a publication that utilizes bladder cancer PDX samplesfrom our tumor bank.
Are NSG™ mice suitable for cancer stem cell studies?
Yes. NSG™ has emerged as the preferred platform for studying the frequency and characteristics of cancer stem cells. This is a consequence of its greater degree of immunodeficiency, and in many instances this results in a more permissive environment for cancer stem cells to grow. Here are examples frommelanoma, leukemia, and many other tumor types. NSG™ mice have enabled some important discoveries, especially for melanoma and acute myeloid leukemia.
Why are NSG™ mice good xenografts hosts for ER+ breast cancer?
Estrogen receptor (ER) positive breast cancers require estrogen (estradiol) supplementation to retain ER positivity in a xenograft setting. NSG™ mice are more resistant to the toxic side effects of estradiol supplementation compared to other strains, including nude and NOD-scid. This enables long-term study of ER+ breast cancer xenografts.
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