An Introduction to Common Immunodeficient Mouse Models

Q: What is the process for establishing human tumor xenografts in immunodeficient mice?

Establishing a human tumor xenograft follows a standardized workflow: 1. Select the host. Choose a strain based on the tumor type and study goal. Options range from T cell-deficient BALB/c nude mice, often enough for CDX, to severely immunodeficient NKG mice (lacking T, B and functional NK cells), which suit difficult-to-engraft PDX, hematologic tumors and humanized models. 2. Prepare the material. For CDX, expand human cancer cells in culture, harvest them in log-phase growth, and resuspend them in serum-free medium, often mixed with a basement membrane matrix such as Matrigel. For PDX, keep fresh patient tumor tissue in cold preservation medium, cut it into small fragments (about 2–3 mm³), and implant it as soon as possible. 3. Inoculate or implant. Inject cells or implant tissue fragments subcutaneously (usually into the flank) for easy monitoring, or orthotopically (into the organ of origin) to better recreate the native tumor microenvironment and metastatic behavior. Hematologic tumors are usually injected intravenously. 4. Monitor and passage. Track tumor growth with calipers or non-invasive imaging such as bioluminescence. Once tumors reach a defined size, passage PDX tumors into new mice and cryopreserve them to build a stable model bank. 5. Validate. Confirm that the xenograft keeps the features of the original tumor with histopathology (H&E and immunohistochemistry) and STR or genomic profiling. Rule out mouse-cell overgrowth and EBV-associated human lymphoma before using the model in efficacy studies.

Q: Why are immunodeficient mice required for establishing human tumor xenograft models?

Immunodeficient mice are needed because of immune rejection. A mouse with an intact immune system recognizes human cells as foreign, and its T cells, supported by B cells, antibodies, NK cells, macrophages and complement, reject the human graft within days to weeks. Strains such as BALB/c nude, NOD-SCID and NKG mice lack some or all of these immune barriers, so human tumor cells can survive, become vascularized by the host, and grow as solid tumors. Researchers can then study the behavior of real human cancers in a living system and test human-specific anti-cancer therapies in vivo before clinical development.

Q: How has the development of immunodeficient mice evolved over the years?

Nude mice (1960s). The story began with the discovery of the athymic nude mouse in 1962. A Foxn1 mutation leaves these mice without a thymus and mature T cells, and in 1969 they hosted the first human tumor xenograft. But they keep functional B cells and highly active NK cells, which limits the human tissues they can accept. SCID mice (1983). A mutation in the Prkdc gene blocks both T and B cell development. However, many SCID mice become “leaky” as they age, producing some functional T and B cells, and their NK activity stays high. NOD-SCID mice (1990s). Moving the scid mutation onto the NOD background added the NOD strain’s innate immune defects, including reduced NK cell, macrophage and complement function, and greatly reduced leakiness. Residual NK activity and a short lifespan caused by thymic lymphomas remained limitations. Il2rg-null mice (2000s). The major breakthrough came from knocking out the IL-2 receptor common gamma chain gene (Il2rg) on the NOD-scid background, first in the NOG (2002) and NSG (2005) strains. This removed functional NK cells. Cyagen's NKG mouse (NOD.Cg-Prkdc^scid Il2rg^em1Cya/Cya) belongs to this generation: it lacks mature T cells, B cells and functional NK cells and is one of the most permissive hosts for human cell and tissue engraftment. Next-generation strains. Newer strains build on this platform. Some express human cytokines, some lack mouse MHC molecules, and some carry Kit mutations, so they better support human immune and hematopoietic cells.

Q: What were the key technical improvements in the latest generation of immunodeficient mice?

Loss of functional NK cells. Knocking out Il2rg (γc/CD132) blocks IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 signaling, which prevents NK cell development. This is the main reason human cell engraftment is so efficient. Reduced innate rejection. The NOD background carries a SIRPα variant that binds human CD47 and lowers macrophage phagocytosis of human cells, along with a complement C5 deficiency. Stability. Leakiness is minimal, so mice rarely develop functional mouse T or B cells as they age. Longevity. Unlike NOD-SCID mice, Il2rg-null strains rarely develop thymic lymphomas and live much longer, which supports long-term studies such as humanized immune system models. Support for humanization. Derivative strains engineered to express human cytokines (e.g., IL-3, GM-CSF, SCF, IL-15) or to lack mouse MHC molecules better support specific human immune cell lineages and reduce GvHD.

Q: Are BALB/c mice considered immunodeficient models?

No. Standard BALB/c mice are immunocompetent, with a complete and functional immune system that includes T cells, B cells and natural killer (NK) cells. The confusion comes from the fact that several popular immunodeficient models are built on the BALB/c background: BALB/c (wild-type). An immunocompetent inbred strain widely used in immunology, antibody production, vaccine research and syngeneic tumor models. Its immune response naturally leans toward a Th2 type. BALB/c nude (Foxn1^nu). An immunodeficient variant. A Foxn1 mutation leaves these mice without a thymus, so they have very few mature T cells. They are also hairless, their most recognizable feature. C.B-17-scid. A BALB/c-congenic strain carrying the scid mutation, lacking both T and B cells.

Cyagen Technical Content Team | June 11, 2025

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Contents

01. Types of Immunodeficient mice

Immunodeficient mice refer to the mice with defects in one or more immune components (such as T, B, NK cells) in the immune system. This type of mouse is widely used in the research of oncology (tumor growth, metastasis, anti-tumor drug screening), immunology (mechanisms of immune cell development and proliferation, pathology of immune diseases), infectious diseases (pathogenic mechanisms of viral/bacterial infectious diseases), and stem cell biology (human stem cell transplantation), and more.

Types of Immunodeficient mice

Nude mice

In 1962, nude mice were first observed by N. R. Grist at Ruchill Hospital in Glasgow and were formally described by Flanagan in 1966. They are called nude mice because they are hairless. In 1968, Pantelouris showed that these mice lack a thymus, making the nude mouse the first recognized immunodeficient mouse model.

Nude mice carry a spontaneous loss-of-function mutation in the Foxn1 gene, which disrupts the development of hair follicles and the thymic epithelium. The resulting athymia and severe deficiency of mature T lymphocytes impair their adaptive immune responses (including T cell-mediated immunity and T helper cell-dependent antibody production). This means that they usually do not reject allografts or xenografts, so nude mice are commonly used as recipients of human tumor xenografts.

However, nude mice still have B cells and NK cells (their NK cells are more active than wild-type mice), and their intact innate immunity limits the engraftment of some human tumors in nude mice. In addition, the number of extrathymically derived T cells increases as nude mice age ("T cell leakiness").

NOD-SCID mice

In 1980, Makino and colleagues (Japan) established the non-obese diabetic (NOD) mouse strain, which spontaneously develops T lymphocyte infiltration of the pancreatic islets (insulitis) and destruction of insulin-producing β cells, a hallmark of autoimmune Type 1 Diabetes. Later studies showed that NOD mice also carry multiple innate immune abnormalities, including deficiency of complement component C5 (and therefore of hemolytic complement activity) and functional defects in NK cells, macrophages, and dendritic cells.

In the early 1990s, researchers at The Jackson Laboratory (Prochazka, Shultz, Leiter et al., 1992) transferred the Prkdc^scid mutation from C.B-17-scid mice onto the NOD background by backcrossing, creating non-obese diabetic (NOD)-severe combined immunodeficiency (SCID) mice; in 1995, Shultz et al. reported their detailed immunological characterization. NOD-SCID mice do not develop diabetes, lack functional T and B cells, and retain the innate immune defects of the NOD background, so they became ideal transplant recipients for human hematopoietic stem cells and human solid tumors.

In addition, Shultz et al. found that, unlike C.B-17-scid mice, in which "leaky" production of functional T and B cells is common and increases with age, NOD-SCID mice show very low leakiness. As such, NOD-SCID mice quickly became an indispensable transplantation model in hematopoietic cell research. Their main limitations are residual NK cell activity and a relatively short lifespan (median ~8.5 months), largely due to a high incidence of spontaneous thymic lymphomas.

NKG mice

The NKG mouse (NOD.Cg-Prkdc^scid Il2rg^em1Cya/Cya) is a severely immunodeficient strain independently developed by Cyagen by deleting the Il2rg gene on the NOD-scid background. Loss of the IL-2 receptor common gamma chain (γc, CD132) blocks signaling by IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, thereby eliminating NK cell development on top of the T and B cell deficiency caused by the scid mutation.

Features:

Deficient in mature T cells, B cells, and functional NK cells
Reduced complement activity (the NOD background carries a C5 complement deficiency)
Macrophage phagocytosis of human cells is weak (NOD-type SIRPα binds human CD47 and delivers a "don't eat me" signal)

NKG mice support highly efficient engraftment of human tumor cell lines (cell line-derived xenografts, CDX) and patient tumor tissues (patient-derived xenografts, PDX). They are also well suited for transplantation of human hematopoietic stem cells (HSC), peripheral blood mononuclear cells (PBMC), and adult stem cells and tissues, making them a suitable host for humanized immune system (HIS) models.

As one of the models with the highest degree of immunodeficiency, NKG mice are widely used in oncology (tumor transplantation and anti-tumor efficacy studies), immunology and tumor immunity, hematopoiesis and blood disorders, infectious diseases (e.g., HIV/AIDS), autoimmune diseases, immunotherapy, vaccine development, GvHD research, and drug safety evaluation.