A Comparative Analysis of Rag2/Il2rg (R2G2) and NSG™ Radiosensitivity

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A comparative analysis of Rag2/Il2rg (R2G2) and NSG™ radiosensitivity.

Introduction The results of an internal The increasing variety of immunodeficient mouse strains that are available study comparing the can make choosing an appropriate model difficult and confusing. This is radiosensitivity of Envigo’s particularly relevant to researchers employing radiation in their studies Rag2/Il2rg double knockout - whether as a pre-conditioning step (e.g. myeloablation) or testing the efficacy of radiation therapy - since different models display inherent (R2G2) model to the NSG™ differences in their sensitivities to radiation. model, confirm that R2G2 Highly immunodeficient strains that carry multiple genetic alterations have mice are less radiosensitive been invaluable to many areas of research. Relevant to this paper are the than NSG™ mice, while both models completely lacking natural killer (NK) cells, which were generated are highly immunodeficient. via deletion or truncation of the common gamma chain/Il2rg (Cao, 1995; DiSanto, 1995; Ohbo, 1996). Since Il2rg is required to mediate the effects of multiple cytokines including IL-2, IL-4, IL-7, IL-9, and IL-15, disruption of the Il2rg gene leads to major defects in lymphocyte and lymphoid tissue development. Mice carrying the Il2rg null allele or truncated mutant have been bred into the non-obese diabetic-severe combined immunodeficiency (NOD.SCID) model to generate the highly immunodeficient NSG™ (Shultz, 2005) and NOG (Ito, 2002) mice, respectively. An equally powerful model is the Rag2/Il2rg double knockout (R2G2) immunodeficient mouse (Goldman, 1998; Mazurier, 1999), a model that is now available from Envigo. In addition to comparable defects in B-, T-, and NK-cells with NSG™/NOG mice, the presence of the Rag2 mutation in place of scid has the benefit of providing a radioresistant phenotype, which is an important feature for a variety of research applications. In addition to providing a comparative summary of Rag2-/- and SCID models, this paper presents the results of a radiosensitivity study which compared the growth and survival of Envigo’s Rag2/Il2rg (R2G2) model to the NSG™ model using three different doses of radiation. With this knowledge in hand, scientists can make better informed decisions when choosing an immunodeficient model for their studies.

North America 800.793.7287 EU and Asia envigo.com/contactus [email protected] envigo.com Comparison of Rag2-/- and SCID models SCID mice and their multigenic counterparts are the most (e.g. Biedermann, 1991; Fulop, 1990; Goldman, 1998; Shultz, commonly used immunodeficient mouse strains. First 2000; Shultz, 2005). Overall, the data support the view that identified in a colony of C.B-17 mice (Bosma, 1983), SCID Rag2 mice are more radioresistant than SCID mouse strains, mice harbor a spontaneous mutation in the Prkdc gene. and help to establish the advantage of the Rag2/Il2rg model Prkdc encodes the catalytic subunit of the DNA-dependent for studies requiring radiation, especially those studies that protein kinase (DNA-PK) and is required for non-homologous may require higher doses of radiation exposure. end joining (NHEJ). Since NHEJ is essential for V(D)J There are two main types of studies in which the recombination, a process which gives rise to immunoglobulin radiosensitivities of immunodeficient mice require careful and T-cell receptor diversity, the mutation of Prkdc results in consideration. First, whole-body irradiation is often carried failed B- and T-cell maturation (Belizario, 2009). out as a pre-conditioning myeloablative step to enhance Unlike the spontaneous mutation carried by SCID mice, the engraftment of transplanted hematopoietic cells (e.g., Rag2-/- animals were engineered to harbor a germline Goldman, 1998). For example, this is a critical step prior to deletion of the recombination-activating gene 2 (Rag2) generating “humanized” immune system mice. Sub-lethal (Mombaerts, 1992; Shinkai, 1992). Since the enzyme product irradiation has been shown to stimulate the bone marrow encoded by this Rag gene serves to ensure proper V(D)J production of stem cell factor (SCF) production and facilitate recombination, its deletion also leads to B- and T-cell hematopoiesis (Broudy, 1997). Irradiation also serves to deficiencies. Notably, the Rag2-/- strain serves as the eliminate endogenous HSCs and other residual immune breeding partner for Il2rg-/- mice to create the Rag2/Il2rg cells to make room for engrafted cells. Notably, the higher double knockout (R2G2). the radiation dose, the greater the likelihood of achieving more complete myeloablation. For instance, Down et al. Although SCID and Rag2-/- mice are often described as used murine bone marrow chimera models to evaluate the exhibiting similar immune dysfunction, the level of B- and efficacy of host total body irradiation given at different doses T-cell function differs between the two models. Indeed, for engraftment of syngeneic and allogeneic bone marrow. SCID mice are known to become “leaky” with age, such that While partial engraftment of syngeneic marrow was seen at virtually all mice more than one-year old contain detectable single doses as low as 2Gy, the donor component became levels of B and T lymphocytes (Nonoyama, 1993), which can increasingly more prominent with increasing radiation dose inhibit xenograft growth. In contrast, Rag2-/- mice do not (100% was achieved at 7Gy). Importantly, resistance of the display a “leaky” phenotype and are thus considered more host appeared to prevent allogeneic engraftment until immunodeficient than SCID mice. This feature of Rag2-/- mice 5.5Gy, and the authors noted that there was a steep radiation also makes them better suited for long-term in vivo assays. dose response observed after that level, so that the level Despite some obvious advantages of the Rag2-/- model over of chimerism ≥6Gy became comparable with syngeneic the SCID model, the SCID model remains more heavily used engraftment (Down, 1991). by researchers. One potential reason for this discrepancy is Of course, investigators must balance the benefits of the fact that the SCID strain was identified in 1983, almost a achieving higher myeloablation with the toxic side effects full decade before advances in recombinant DNA technology of radiation by considering factors such as the strain and led to the generation of Rag2-/- animals. During this time, age of recipient mice. Second, the radiosensitivities of SCID mice were made widely available to researchers around immunodeficient mice are also an important factor to the world who, in turn, generated reliable data regarding the consider for oncology studies. Along with chemotherapy, immunological properties of the model and optimization of radiation therapy remains an integral part of anti-cancer protocols for human cell transplantation. Nevertheless, as treatment regimens with up to 75% of patients requiring the interest and use of immunodeficient mice continues to some form of radiation during the course of treatment rise, scientists are becoming more aware of the advantages (Kahn, 2012). In addition to the direct effects of radiation offered by other models, including Rag2-/- mice. on the tumor, there are also bystander effects (also known Radiosensitivities of Rag2-/- and SCID models as abscopal effects) induced by radiation (mostly at higher doses). These effects are thought to be immune-mediated In addition to having a “leaky” immunodeficient phenotype, and their implications for cancer therapy are only beginning the SCID mouse is known for being hypersensitive to to be unraveled (Sologuren, 2014; Demaria, 2004). ionizing radiation (Biedermann, 1991; Fulop, 1990). As noted earlier, NHEJ plays multiple roles in cells. It is required for On this note, in a study by Shiraishi et al., it was shown V(D)J recombination, as well as for proper DNA repair. that the administration of a chemokine after local tumor Consequently, the disruption of NHEJ through the mutation site irradiation (using a dose of 6Gy) prolonged survival of of the Prkdc gene renders SCID mice sensitive to DNA the mice. Indeed, the tumor was completely eradicated in damage induced by radiation exposure. Furthermore, as the about 50% of the animals with daily administration of the mutation is present in every cell, all tissues have the potential chemokine, and importantly, tumor growth at the non- to be affected. In contrast, the radiosensitivity of Rag2 mice irradiated site was inhibited, suggesting the chemokine is comparable to wild-type mice, as the Rag2 enzyme is only enhanced the bystander effect of radiation (Shiraishi, 2008). required for V(D)J recombination. Several studies have tested While mouse-based radiation therapy studies certainly the radiosensitivity of immunodeficient mouse strains

North America 800.793.7287 EU and Asia envigo.com/contactus [email protected] envigo.com exist in the literature (e.g., Speers, 2016; Karnak, 2014; Results Shiraishi, 2008), advances in technology, such as small The effect of varying doses of radiation on the survival animal image-guided radiation platforms (Butterworth, and body weight of R2G2 and NSG™ mice was evaluated. 2015), will undoubtedly contribute to a rise in the use of Groups of mice were irradiated with radiation doses of 2Gy, radiation therapy in pre-clinical studies in the near future. 4Gy, or 6Gy and monitored for 29 days. As shown in Figures The availability of radioresistant mice including the R2G2 1A and 2A, no meaningful differences in survival or body model will allow scientists to implement these advanced weight change were observed between the R2G2 and NSG™ technologies into their research. models at the lowest dose of radiation (2Gy). Survivability Overall, researchers need to carefully consider was 100% for both models at 29 days (Figure 1A), and after immunodeficient model selection for their studies. an initial small drop in body weight in both models, after 29 days the R2G2 and NSG™ mice had gained an average This can be a complex decision, as many factors are involved of 5.1% and 8.8%, respectively (Figure 2A). In contrast, at and many strains of immunodeficient mice are available. a radiation dose of 4Gy, NSG™ mice experienced 100% When a model with enhanced radioresistance is warranted, mortality by post-irradiation day 8 (Figure 1B), and showed it is advisable to choose a Rag-based model, such as the a drastic loss of body weight (Figure 2B). Meanwhile, R2G2 highly immunodeficient R2G2, which is a suitable host for mice displayed 100% survivability (Figure 1B), and robust xenografts using conventional immortalized cancer cell lines body weight gain showing a similar trend to that of the 2Gy and patient-derived tissues or cells, and is also ideal for radiation dose (Figure 2B). At the highest radiation dose of studies examining the efficacy of radiation as part of a 6Gy, approximately 50% of the R2G2 mice survived for 14 cancer treatment regimen. days, but by the 17-day timepoint, there was 100% mortality In the following section we show that Envigo's R2G2 is more (Figure 1C). For the NSG™ mice, these animals experienced radioresistant than the NSG mouse, using three different 100% mortality by the 5-day timepoint (Figure 1C). Thus, with doses of radiation. regard to survivability, the R2G2 mice survived approximately three-fold longer than the NSG™ mice following a radiation Radiosensitivity study dose of 6Gy. As for body weight, both models exhibited A study was conducted using Envigo’s R2G2 model (B6;129- reduced body weight after the 6Gy dose of radiation, albeit Rag2tm1FwaII2rgtm1Rsky /DwlHsd) to empirically characterize its the body weight loss of the R2G2 mice was gradual, in radiosensitivity. For comparative purposes, NSG™ (NOD. contrast to the rapid body weight loss seen in the NSG™ Cg-Prkdcscid Il2rgtm1Wjl /SzJ) mice were also included, and this mice (Figure 2C). model has been shown to tolerate radiation doses of up to Overall, the results of the radiosensitivity study demonstrate 4Gy (Shultz, 2005). A summary of the methods and results of that R2G2 mice are less radiosensitive at radiation doses of the study are presented below. 4Gy and 6Gy, relative to NSG™ mice. Indeed, even at the Materials and methods highest dose of radiation tested (6Gy), R2G2 mice showed 50% survivability at 14 days, and only a gradual loss of body On study day 1, groups of R2G2 and NSG™ mice (n=5 per weight over the time-period leading up to 100% mortality group) aged 7-8 weeks were exposed to one of three doses of these animals. This contrasts with the 100% mortality of whole body irradiation: 2Gy, 4Gy or 6Gy. Mice were observed for NSG™ mice after only five days, which was irradiated using a RadSource RS-2000, which employs a 160 accompanied by dramatic body weight loss. kV, 4.2 kW x-ray source. After receiving the radiation dose, animals were monitored daily for changes in body weight and survival for up to 30 days. Animals were euthanized when moribund (e.g. >20% body weight loss, loss of righting reflex). All animals were maintained on Teklad Global Rodent Diet 2918 (18% protein).

North America 800.793.7287 EU and Asia envigo.com/contactus [email protected] envigo.com Figure 1. Percent Survival of Envigo’s R2G2 model and NSG™ mice irradiated at 2Gy (A), 4Gy (B), and 6Gy (C) (Green = R2G2; Dark grey = NSG™).

Figure 1A: Percent Survival of Envigo’s R2G2 model and NSG™ mice irradiated at 2Gy

100 Radiation dose given on study day 1

90 Maintained on Teklad Global Rodent Diet 2918 80 (18% Protein)

40 Percent survival

10 R2G2 -2Gy

NSG-2Gy 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Study day

Figure 1B: Percent Survival of Envigo’s R2G2 model and NSG™ mice irradiated at 4Gy

100 Radiation dose given on study day 1

90 Maintained on Teklad Global Rodent Diet 2918 80 (18% Protein)

Percent survival 40

10 R2G2 -4Gy

NSG-4Gy 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Study day

North America 800.793.7287 EU and Asia envigo.com/contactus [email protected] envigo.com Figure 1C: Percent Survival of Envigo’s R2G2 model and NSG™ mice irradiated at 6Gy

100 Radiation dose given on study day 1

90 Maintained on Teklad Global Rodent Diet 2918 80 (18% Protein)

30 Percent survival

10 R2G2 -6Gy

NSG-6Gy 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Study day

North America 800.793.7287 EU and Asia envigo.com/contactus [email protected] envigo.com Figure 2. Percent change in body weight of Envigo’s R2G2 model and NSG™ mice irradiated at 2Gy (A), 4Gy (B), and 6Gy (C) (Green = R2G2; Dark grey = NSG™).

Figure 2A: Percent change in body weight of Envigo’s R2G2 model and NSG™ mice irradiated at 2Gy

20.0 Radiation dose given on study day 1

Data represented are average ± SD 15.0 Maintained on Teklad Global Rodent Diet 2918 (18% Protein) 10.0

5.0

0.0 Body weight (percent change)

-5.0

R2G2 -2Gy

NSG-2Gy -10.0 1 2 3 4 5 8 9 10 11 12 15 16 17 18 19 22 23 24 25 26 29

Study day

Figure 2B: Percent change in body weight of Envigo’s R2G2 model and NSG™ mice irradiated at 4Gy

10.0 Radiation dose given on study day 1

Data represented are 5.0 average ± SD

Maintained on 0.0 Teklad Global Rodent Diet 2918 (18% Protein)

-5.0

-10.0

-15.0 Body weight (percent change) -20.0

-25.0 R2G2 -4Gy

NSG-4Gy -30.0 1 2 3 4 5 8 9 10 11 12 15 16 17 18 19 22 23 24 25 26 29

Study day

North America 800.793.7287 EU and Asia envigo.com/contactus [email protected] envigo.com Figure 2C: Percent change in body weight of Envigo’s R2G2 model and NSG™ mice irradiated at 6Gy

5.0 Radiation dose given on study day 1

Data represented are average ± SD 0.0 Maintained on Teklad Global Rodent Diet 2918 (18% Protein) -5.0

-10.0

-15.0 Body weight (percent change)

-20.0

R2G2 -6Gy

NSG-6Gy -25.0 1 2 3 4 5 8 9 10 11 12 15 16 17 18 19 22 23 24 25 26 29

Study day

Conclusions The development of highly immunodeficient strains of mice radioresistance than SCID strains. Thus, for scientists has ushered in an era of advanced research models. However, pursuing studies that require an immunodeficient model as touched upon in this paper, not all immunodeficient and exposure to higher doses of irradiation, the R2G2 mice are equal, and understanding the inherent differences model is the superior choice. associated with each strain is critical when planning a study, A wide variety of options are available when it comes to especially those requiring irradiation. The results of an choosing an immunodeficient mouse, and model selection internal study comparing the radiosensitivity of Envigo’s is not always straightforward. Discussions with Envigo Rag2/Il2rg double knockout (R2G2) model to the NSG™ personnel when planning a project can help save time, model, confirm that R2G2 mice are less radiosensitive than effort, and resources. NSG™ mice, while both are highly immunodeficient. The R2G2 mouse features a higher degree of immunodeficiency than many models (e.g. athymic nude), and greater

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