Below are common questions and answers for maintaining and using NSG™ mice in biomedical research. The questions are organized under the following sections:
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NSG™ is one of the most immunodeficient mouse strains described to date. Here's why:
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).
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.
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 thePrkdcscidmutation 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.
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.
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 thanscid-beige. scid beige mice are not available from The Jackson Laboratory.
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:
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.
NSG™ and NRG (NOD.Cg-Rag1tm1MomIl2rgtm1Wjl/SzJ, ) 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.
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.
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.
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.
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.
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).
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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.
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.
Yes, through In Vivo Pharmacology Services.
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.
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:
Other analyses of the different lineages can be found in the online categorized list of references.
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.
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.
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-PrkdcscidIl2rgtm1Wjl Tg(HLA-A2.1)1Enge/SzJ (Stock # ) and NOD.Cg-PrkdcscidIl2rgtm1Wjl Tg(HLA-A/H2-D/B2M)1Dvs/SzJ (Stock # ) 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-PrkdcscidIl2rgtm1Wjl Tg(HLA-DRA*,HLA-DRB1*)1Dmz/GckRolyJ (Stock # ) and NOD.Cg-PrkdcscidIl2rgtm1Wjl H2-Ab1tm1Gru Tg(HLA-DRB1)31Dmz/SzJ (Stock # ) are two examples. The second strain does not express the endogenous mouse MHC class2 complex.
No. Only hematopoietic stem cells require preconditioning irradiation for efficient engraftment. Irradiation accelerates the GVHD response.
Below is a list of publications that describe protocols and considerations for creating humanized NSG™ mice. Pearson, et al. is especially recommended.
Certain cancer models are established more efficiently in NSG™ mice compared to other strains:
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. Lung and ovarian PDX models are described in the literature, as is a publication that utilizes bladder cancer PDX samples from our tumor bank.
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 from melanoma, leukemia, and many other tumor types. NSG™ mice have enabled some important discoveries, especially for melanoma and acute myeloid leukemia.
Estrogen receptor (ER) positive breast cancers require estrogen (estradiol) supplementation to retain ER positivity in a xenograft setting. NSG™ mice are resistant to the toxic side effects of estradiol supplementation, enabling long-term study of ER+ breast cancer xenografts.
Here is an A-Z list of immunology tests, together with a brief summary of the clinical applications of each. This is intended purely as a guide.
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