Scid mutants or Rag2 Knockouts - Which Immunodeficient Model is Best for Your Study?

Mice with compromised immune systems are invaluable research tools. Starting with the discovery of athymic nude mice in the 1960's, the portfolio of immunodeficient rodents has grown significantly. Although these models are used quite widely, not all researchers understand their intrinsic characteristics and how those characteristics impact experimental outcomes. Learn more about two key immunodeficient models, scid mutants and Rag2 knockouts, and how to choose the most appropriate model for your study.

The scid Mutation

The scid mutation was first discovered in 1983 by Mel Bosma at the Fox Chase Cancer Center1. Scid stands for severe combined immunodeficiency. This mutation arose spontaneously in a colony of C.B-17 mice that Bosma was using for studies of immunoglobulin isotypes. The scid mutation is a mutation in the Prkdc gene, protein kinase, DNA activated, catalytic polypeptide. The specific genetic alteration in the Prkdcscid allele is an T-to-A transversion point mutation at codon 4095, which creates a premature stop codon2,3. Prkdc is involved in DNA repair via non-homologous end joining (NHEJ). V(D)J recombination, which rearranges the genetic components of antibodies and T cell receptors during B and T cell development, requires NHEJ for proper function.

Due to the NHEJ defect in Prkdcscid mutants, scid mice lack both mature T and B cells. As one of the first immunodeficient models available, that original scid, the C.B-17 scid, was disseminated worldwide and has been used in thousands of studies. The Prkdcscid allele was also backcrossed onto other strain and stock backgrounds to make models such as the outbred ICR scid and the NOD scid. Among the scids that Taconic Biosciences offers, the C.B-17 scid is the most requested, primarily due to the wealth of historical data and publications using the strain. But it may not always be the best experimental choice.

Development of some functional T and B cells

Scid mice can become "leaky", meaning that some functional T and B cells can develop. This appears to arise from a low frequency of V(D)J coding joint formation, with recombination often occurring at sections of short homology5. This leakiness varies by the strain/stock background, with age and by housing conditions, with the original C.B-17 scid actually being the leakiest strain.
Leakiness comparison of NOG, NOD-scid and C.B-17 scid
IgG+M antibody levels in the sera from aged NOG (7-10 months old) and NOD-scid (6-7 months old) mice were measured by ELISA. In C.B-17- scid (6-9 months old) mice, serum IgG+M+A antibody levels measured in 1989 were used in this figure.

While the C.B-17 scid may be fine for short term experiments, longer term experiments may be challenging. The ICR scid is a less leaky alternative on an outbred background. As an outbred, ICR scid mice are larger, hardier and less expensive. However, the outbred background can introduce more experimental variation. Note that larger animals also require additional compound for dosing. In general, the ICR scid is an excellent value. The NOD scid is special in that the NOD inbred strain background imparts some additional immunodeficiency. NOD mice lack complement C5, and they have defects in macrophage and dendritic cell function. They are also on the less leaky end of the spectrum. This makes it the most immunocompromised of the basic scid models. The major drawback to the NOD scid is that it develops thymic lymphomas at a high rate, so the effective lifespan is somewhere between 7-9 months. Longer term studies are not advisable in the NOD scid strain.

The scid mutation introduces a few other special considerations. As this mutation affects DNA repair, mice carrying Prkdcscid are more sensitive to radiation. They are thus not ideal choices for experiments involving clinically relevant radiation doses. Scid mice may also be more sensitive to drugs which damage DNA. When using drugs with this type of mechanism, a tolerance study is recommended to help choose appropriate doses for follow on efficacy studies.

Knockout of the Rag2 gene

The basic mechanism of V(D)J recombination.
Adapted from Nature Reviews Genetics.
Rag2 knockouts are somewhat lesser known compared to scid mice but offer numerous advantages. The Rag2 gene, recombination activating gene 2, was knocked out in 1992 by Frederick Alt at Columbia University6. The Rag2 protein is also involved in V(D)J recombination. Both Rag2 and Rag1 bind DNA at recombination signal sequences and create single strand nicks as the first step of recombination. Arresting this pathway blocks maturation of T and B cells4. So the immunodeficient phenotype of Rag2 knockouts is similar to that of scid mice. One big difference is that the Rag2 mutation is not leaky. Knocking out Rag1 or Rag2 means that V(D)J recombination cannot be initiated, so no leakiness occurs through downstream successful recombination events as in the scid. The non-leaky phenotype of Rag2 knockout mice allows longer term experiments without worry of increased graft rejection as the mice age. Rag2 mice also tolerate higher radiation doses compared to scid mice. The Rag2 mutation has been backcrossed to a number of backgrounds, primarily inbred strains, as well as combined with other mutations to make a number of useful models both for oncology as xenograft hosts as well as in basic immunology research. Rag2 knockout models tend to be used quite widely in adoptive transfer experiments, both due to the availability on key strain backgrounds such as B6 and BALB/c as well as to their non-leaky nature.

So why isn't the Rag2 used as widely as the scid? Much of this can be traced to historical accident and the weight of the publication record. The scid got a head start! But researchers are starting to realize the advantages of the Rag2 knockout and choose it for experiments where a scid might have drawbacks. Use of Rag2 knockouts has increased significantly, and that trend is likely to continue. For your next experiment, consider carefully the unique characteristics of these two models and determine which best meets your needs.

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1. Bosma GC, Custer RP, Bosma MJ. (1983) A severe combined immunodeficiency mutation in the mouse. Nature. 301(5900):527-30.
2. Blunt T, Gell D, Fox M, Taccioli GE, Lehmann AR, Jackson SP, Jeggo PA. (1996) Identification of a nonsense mutation in the carboxyl-terminal region of DNA-dependent protein kinase catalytic subunit in the scid mouse. Proc Natl Acad Sci U S A. 93(19):10285-90.
3. Araki R, Fujimori A, Hamatani K, Mita K, Saito T, Mori M, Fukumura R, Morimyo M, Muto M, Itoh M, Tatsumi K, Abe M. (1997) Nonsense mutation at Tyr-4046 in the DNA-dependent protein kinase catalytic subunit of severe combined immune deficiency mice. Proc Natl Acad Sci U S A. 94(6):2438-43.
4. Lieber MR. (2010) The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annu Rev Biochem. 79:181-211.
5. Kotloff DB, Bosma MJ, Ruetsch NR. (1993) V(D)J recombination in peritoneal B cells of leaky scid mice. J Exp Med. 178(6):1981-94.
6. Shinkai Y, Rathbun G, Lam KP, Oltz EM, Stewart V, Mendelsohn M, Charron J, Datta M, Young F, Stall AM, et al. (1992) RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell. 68(5):855-67.

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