RAG1-knockout mice, commonly written as Rag1⁻/⁻, lack the gene responsible for assembling the receptors that B cells and T cells need to recognize threats, leaving these animals without functional adaptive immunity. They develop normally in most other respects, which makes them one of the most widely used immunodeficient mouse models in biomedical research. But the story of RAG1 mice extends well beyond missing lymphocytes. Researchers have uncovered unexpected roles for the RAG1 gene in the brain, documented striking behavioral changes in these animals, and used them as living platforms for studying everything from cancer xenografts to gut-brain signaling.
Why RAG1 Matters for the Immune System
The RAG1 protein works in tandem with RAG2 to carry out a process that shuffles segments of DNA in developing lymphocytes, generating the vast diversity of antibodies and T cell receptors that vertebrates rely on to fight infections. Without this gene-segment rearrangement, neither B cells nor T cells can mature past an early developmental stage.1PubMed Central. RAG: a recombinase diversified In the early 1990s, researchers generated mice with a targeted disruption of the RAG1 gene and confirmed that these animals have small lymphoid organs containing no mature B or T lymphocytes.2Cell. RAG-1-deficient mice have small lymphoid organs that do not contain mature B and T lymphocytes The block occurs early in lymphocyte development and tracks directly to the inability to rearrange antigen receptor genes.
This clean, well-characterized immune deficit is what makes Rag1⁻/⁻ mice so valuable. Unlike mice treated with drugs that suppress immunity broadly, the RAG1 knockout removes adaptive immunity at its genetic root while leaving other organ systems largely intact. Researchers get a predictable baseline: no antibodies, no T cell responses, but a functioning innate immune system, normal organ development, and a lifespan long enough for chronic experiments.
What Innate Immunity Looks Like Without Adaptive Backup
Losing B and T cells does not leave Rag1⁻/⁻ mice defenseless. Their innate immune system, including natural killer cells, macrophages, and innate lymphoid cells, remains active. In some cases it overcompensates. Natural killer cells in Rag1⁻/⁻ mice appear comparable in number and function to those in normal mice.3PubMed Central. Natural Killer Cells Are Present in Rag1(-/-) Mice and Promote Tissue Damage During the Acute Phase of Ischemic Stroke In a stroke model, these NK cells actually promoted tissue damage: when researchers depleted NK cells with an antibody, the Rag1⁻/⁻ mice developed smaller brain infarctions and better behavioral scores. That finding serves as a reminder that studies using Rag1⁻/⁻ mice to investigate immune cell transfer need to account for the NK cells already present.
A similar theme of innate expansion shows up with group 2 innate lymphoid cells. In RAG-deficient mice, these cells expand and produce elevated levels of inflammatory signaling molecules at baseline, contributing to increased inflammation in skin disease models resembling atopic dermatitis.4PubMed Central. RAG suppresses group 2 innate lymphoid cells The implication is that RAG expression in healthy animals normally restrains certain innate populations. Remove it, and those populations can overshoot, creating an inflammatory environment that researchers need to factor into their experimental design.
How Rag1⁻/⁻ Mice Handle Infections
Without adaptive immunity, Rag1⁻/⁻ mice can mount initial innate responses to pathogens but struggle to clear infections over time. This makes them useful for dissecting exactly which arm of the immune system controls a given pathogen. Work with the bacterium Brucella melitensis illustrates the pattern. Normal mice cleared bacteria from their lungs within about 8 to 12 weeks and kept infection in the liver and spleen at low levels. Rag1⁻/⁻ mice failed to reduce bacterial counts in any organ. In the spleen, bacterial numbers climbed steadily from 2 to 4.5 log units and plateaued, while liver counts continued rising for up to 20 weeks.5PubMed Central. Impaired control of Brucella melitensis infection in Rag1-deficient mice The takeaway was that innate immunity alone can hold Brucella in check at a high set-point in the spleen but cannot control the liver at all; clearance depends on T and B cells.
RAG1-knockout mice have also been used as hosts for human immune cells to study viral infections that do not naturally infect mice. In humanized Rag1 mice reconstituted with human tissue, the retrovirus HTLV-1 established detectable infection in the blood within two weeks, with sustained viral loads and eventual infiltration of immune cells into the central nervous system alongside myelin disruption.6PubMed Central. HTLV-1 Infection and Neuropathogenesis in the Context of Rag1−/−γc−−/− (RAG1-Hu) and BLT Mice This kind of humanized-mouse model is one of the few ways researchers can study human-specific pathogens in a living system.
Behavioral Changes That Caught Researchers Off Guard
If you think of a gene knockout as affecting only one system, RAG1 mice deliver a surprise. Multiple research groups have documented altered behavior in these animals, spanning anxiety, repetitive actions, and memory. Rag1⁻/⁻ mice show significantly increased digging and marble-burying compared to normal mice, behaviors often interpreted as repetitive or compulsive-like in rodent models.7PubMed Central. CD4⁺ but not CD8⁺ T cells revert the impaired emotional behavior of immunocompromised RAG-1-deficient mice They also display altered baseline anxiety and memory, along with an overactive stress-hormone axis and reduced activity in the hippocampus, a brain region central to memory formation.8PubMed Central. Probiotics normalize the gut-brain-microbiota axis in immunodeficient mice
Social recognition memory is another area where Rag1⁻/⁻ mice differ from controls. When tested for their ability to distinguish a familiar mouse from an unfamiliar one, Rag1-deficient animals performed poorly. Intriguingly, Rag2-deficient mice, which have the same immunodeficiency but a different gene knocked out, did not share this specific deficit. Because both strains lack mature lymphocytes, the social memory impairment in Rag1⁻/⁻ mice does not seem to be a simple downstream effect of having no adaptive immune system. The RAG1 gene itself appears to play some direct role.9PubMed Central. Impaired social recognition memory in recombination activating gene 1-deficient mice The mice habituated normally to non-social odors and to open-field environments, suggesting the problem is specific to social memory rather than a general failure to process novelty.
The picture for spatial learning is less clear-cut. One study in rats found that knocking down RAG1 in the hippocampus impaired performance in a water maze spatial learning task.10PubMed. Contribution of Rag1 to spatial memory ability in rats But an earlier study in Rag1-knockout mice found no significant differences in spatial learning and memory, with only modest differences on a visible-platform version of the maze.11PubMed Central. Neurobehavioral changes resulting from recombinase activation gene 1 deletion These conflicting results may reflect differences in species, testing protocols, or whether the gene was absent from birth versus silenced in adulthood. Regardless, the broader theme holds: losing RAG1 changes brain function in ways that go beyond what you would expect from immune deficiency alone.
Can Immune Cells Rescue the Brain?
One of the most striking lines of evidence connecting immunity to behavior comes from adoptive transfer experiments, where researchers inject immune cells into Rag1⁻/⁻ mice and check whether behavioral deficits reverse. When CD4-positive T cells were transferred back into Rag1⁻/⁻ mice, the increased marble-burying and digging behavior reverted toward normal levels. CD8-positive T cells, by contrast, did not produce the same rescue.7PubMed Central. CD4⁺ but not CD8⁺ T cells revert the impaired emotional behavior of immunocompromised RAG-1-deficient mice This specificity suggests that CD4 T cells in particular contribute something to normal emotional regulation in the brain.
Spatial learning deficits in immunodeficient mice can similarly be rescued by transferring T cells. Research using multiple transgenic models demonstrated that an antigen-specific CD4 T cell population is needed for normal performance on the Morris water maze, a standard test of spatial learning and memory.12PubMed Central. Brain antigen-reactive CD4+ T cells are sufficient to support learning behavior in mice with limited T cell repertoire The T cells that mattered recognized brain-associated antigens, hinting at a feedback loop where the immune system monitors the brain and supports its function.
Related work using Rag2-knockout mice (which share the adaptive immune deficit) showed that neonatal lymphocyte reconstitution could restore locomotor activity in adolescent animals, though anxiety-like behavior in the open field test was not rescued.13PubMed Central. Neonatal adoptive transfer of lymphocytes rescues social behavior during adolescence in immune deficient mice The timing of reconstitution seems to matter: some behavioral windows may close if the immune system is not present during critical developmental periods.
RAG1 Expression in the Brain
The behavioral findings become less mysterious when you learn that RAG1 is not exclusively an immune gene. Its transcript was detected in the murine central nervous system as far back as the early 1990s, widespread in both embryonic and postnatal neurons. Expression was most prominent in brain regions with high neuronal density, particularly the cerebellum and the hippocampal formation.14Cell. The recombination activating gene-1 (RAG-1) transcript is present in the murine central nervous system The original investigators speculated that RAG1 might recombine elements of the neuronal genome or protect against detrimental DNA alterations in these long-lived cells.
During mouse brain development, RAG1 expression follows a pattern tied to neurogenesis, appearing first in proliferative zones and then spreading to the developing cortical plate.15PubMed. Expression of RAG-1 in brain during mouse development RAG1 expression has even been found outside mammals: in zebrafish, it shows up in a subset of olfactory sensory neurons, though knocking it out did not disrupt the targeting of olfactory axons or the detection of amino acid odorants.16PubMed Central. The recombination activation gene 1 (Rag1) is expressed in a subset of zebrafish olfactory neurons but is not essential for axon targeting or amino acid detection The precise function of RAG1 in neurons remains unresolved, but its conserved expression across species and developmental stages suggests it is doing something beyond immune gene assembly.
This dual expression helps explain why Rag1⁻/⁻ and Rag2⁻/⁻ mice sometimes behave differently despite sharing the same immune deficit. If RAG1 has a neuron-intrinsic role that RAG2 does not share, then knocking out RAG1 removes both immune function and a brain function, while knocking out RAG2 removes only the immune component. That dissociation has become an important experimental tool for separating immune-mediated behavioral effects from gene-intrinsic ones.
The Gut-Brain-Microbiota Connection
The absence of adaptive immunity in Rag1⁻/⁻ mice also reshapes their gut. Without antibodies to help regulate microbial communities, these mice develop an altered intestinal microbial profile, increased intestinal permeability, and heightened ion transport in the colon. Their stress-hormone axis becomes overactive, and hippocampal signaling is dampened.8PubMed Central. Probiotics normalize the gut-brain-microbiota axis in immunodeficient mice When these mice were treated with probiotics containing Lactobacillus species, intestinal permeability decreased, the microbial community shifted toward a healthier profile, and stress-axis function improved.17PubMed Central. Gut microbiota’s effect on mental health: The gut-brain axis
This finding established Rag1⁻/⁻ mice as one of the clearer demonstrations that the adaptive immune system plays a role in maintaining the gut-brain axis.18Nutrition Reviews. Relationship between the gut microbiome and brain function It also introduced a wrinkle for anyone studying behavior in these animals: some of the behavioral changes attributed to missing lymphocytes may be partially mediated through gut dysbiosis rather than through direct immune-brain signaling. Disentangling the two pathways remains an active challenge.
Cancer Research and Tissue Transplantation
Rag1⁻/⁻ mice are workhorses in cancer biology. Because they cannot reject foreign tissue, they readily accept grafts of human tumor cells, allowing researchers to grow and study human cancers in a living animal. They have been used as hosts for human breast cancer, leukemia, and mouse glioma cell lines to test whether genetically diverse immunodeficient backgrounds can support tumor engraftment.19PubMed Central. Genetically diverse mouse platform to xenograft cancer cells Compared to some other immunodeficient strains, Rag1 knockouts offer a balance: enough immune suppression for grafts to take, but enough residual innate immunity to preserve aspects of the tumor microenvironment.
The model extends beyond cancer. In dermatology research, human skin grafted onto Rag1⁻/⁻ mice was used to study the effects of chronic ultraviolet B exposure. The grafts developed benign cystic tumors and squamous cell carcinomas under UV light, and the researchers concluded that Rag1-deficient mice were superior to the older SCID mouse model for this kind of chronic UV study.20PubMed. UVB induction of epithelial tumors in human skin using a RAG-1 mouse xenograft model The advantage of the Rag1 knockout over SCID mice partly comes from a cleaner genetic background and more predictable immune phenotype.
Methodological Pitfalls and Considerations
As widely used as Rag1⁻/⁻ mice are, interpreting results from them requires care. One recurring issue is the assumption that any phenotype observed in these mice reflects the absence of adaptive immunity. As the behavioral data demonstrate, that assumption breaks down when the knocked-out gene has functions outside the immune system. Comparing Rag1⁻/⁻ with Rag2⁻/⁻ mice is one way to control for this, but it is not always done.
Another concern involves innate immune compensation. The expanded NK cell and innate lymphoid cell populations described earlier can confound experiments that use Rag1⁻/⁻ mice as “immune-deficient” controls. In the stroke study, for example, conclusions about whether adaptive immunity protects or harms the brain during ischemia would have been wrong if the investigators had ignored the NK cells already present in the knockout animals.3PubMed Central. Natural Killer Cells Are Present in Rag1(-/-) Mice and Promote Tissue Damage During the Acute Phase of Ischemic Stroke
Background strain also matters. Studies using Rag1⁻/⁻ mice on different genetic backgrounds have sometimes produced different results for the same experimental protocol. In renal ischemia-reperfusion injury, for instance, Rag1-deficient mice from two different background strains were not protected from kidney damage, complicating the interpretation of whether adaptive immunity drives injury in that context.21PubMed. Effects of combined T- and B-cell deficiency on murine ischemia reperfusion injury Strain effects can interact with the knockout in unpredictable ways, and results from one Rag1⁻/⁻ colony do not always generalize to another.
Adoptive transfer experiments, a major use case for these animals, carry their own caveats. When naive T cells are transferred into Rag1⁻/⁻ hosts, the chronically lymphopenic environment triggers rapid proliferative responses driven by antigens from gut bacteria rather than by the intended experimental antigen. This spontaneous proliferation can alter the tempo and intensity of the T cell response being studied.22PubMed Central. Spontaneous Proliferation of CD4+ T Cells in RAG-Deficient Hosts Promotes Antigen-Independent but IL-2-Dependent Strong Proliferative Response of Naïve CD8+ T Cells The effect disappears when the same transfer is done in germ-free Rag1⁻/⁻ mice, pinpointing gut microbes as the drivers.
Hypomorphic RAG1 Mutations and Partial Immune Function
Not every RAG1 mutation produces a total knockout. Some mutations reduce the protein’s activity without eliminating it entirely, creating what immunologists call hypomorphic alleles. Researchers have used CRISPR to generate mice carrying the same hypomorphic Rag1 mutations found in human patients with combined immunodeficiency and autoimmune features. These mice develop some T and B cells but with a heavily skewed repertoire. They produce serum antibodies yet mount poor responses to immunization and generate autoantibodies, closely mirroring the human disease.23Blood. Hypomorphic Rag1 mutations alter the preimmune repertoire at early stages of lymphoid development These partially functional models fill a gap that complete knockouts cannot: they let researchers study what happens when the immune repertoire is narrowed rather than absent, which more closely resembles conditions in actual human patients.
RAG1 and Rag2 in Aging Brains
Immunodeficient mice are increasingly used to study transplanted human cells in aging brains, and here the choice between Rag1 and Rag2 knockouts turns out to matter more than researchers initially appreciated. Rag2⁻/⁻ brains accumulate lipofuscin, an autofluorescent pigment associated with cellular aging, at much earlier ages and in broader distribution than Rag1⁻/⁻ brains.24Stem Cell Reports. Rag2(-/-) accelerates lipofuscin accumulation in the brain: Implications for human stem cell brain transplantation studies The lipofuscin shows up in various glial cells, including transplanted human microglia, and its autofluorescence can confound imaging studies that rely on fluorescent reporters. For researchers planning long-term brain transplantation experiments in aged immunodeficient mice, this difference between the two knockout strains has direct practical consequences for which model to choose.
The Evolutionary Backstory of RAG1
RAG1’s importance goes far beyond any single mouse model. The gene traces its ancestry to an ancient mobile genetic element, a type of “jumping gene” called a transposon. The catalytic core of RAG1 evolved from the transposase enzyme of the Transib transposon superfamily, and both RAG1 and RAG2 appear to have originated from the same transposable element.25PubMed Central. Evolution of the RAG1-RAG2 locus: both proteins came from the same transposon The strongest evidence for this came from the discovery of ProtoRAG, a DNA transposon family found in lancelets, which are among the most ancient living relatives of vertebrates. ProtoRAG contains both RAG1 and RAG2 gene homologs flanked by the hallmarks of an active transposon.26PubMed Central. Discovery of an Active RAG Transposon Illuminates the Origins of V(D)J Recombination
At some point roughly 500 million years ago, this parasitic DNA element was domesticated by an ancestor of jawed vertebrates and repurposed to shuffle gene segments in developing lymphocytes. That single co-option event gave rise to the entire system of antibody and T cell receptor diversity that defines adaptive immunity in sharks, fish, reptiles, birds, and mammals. The RAG1-knockout mouse, in a sense, reverses that evolutionary milestone: it shows what a vertebrate looks like when the ancient transposon’s legacy is erased from the genome.