Generation and characterization of RAG2 knockout pigs as animal model for severe combined immunodeficiency

Highlights

• We successfully generated RAG2 knockout pigs by somatic cell nuclear transfer.

• RAG2 knockout pigs lacked T and B cells but not NK cells.

• RAG2 knockout pigs had primordial lymph nodes and hypoplastic thymi.

• RAG2 knockout pigs had cells resembling progenitors of T and B cells.

• IL2RG/RAG2 knockout pigs lacked T, B, and NK cells altogether.

Abstract

Pigs with severe combined immunodeficiency (SCID) are versatile animal models for human medical research because of their biological similarities to humans, suitable body size, and longevity for practical research. SCID pigs with defined mutation(s) can be an invaluable tool for research on porcine immunity. In this study, we produced RAG2-knockout pigs via somatic cell nuclear transfer and analyzed their phenotype. The V(D)J recombination processes were confirmed as being inactivated. They consistently lacked mature T and B cells but had substantial numbers of cells considered to be T- or B-cell progenitors as well as NK cells. They also lacked thymic medulla and lymphoid aggregations in the spleen, mesenteric lymph nodes, and ileal Peyer’s patches. We showed more severe immunological defects in the RAG2 and IL2RG double-knockout pig through this study. Thus, SCID pigs could be promising animal models not only for translational medical research but also for immunological studies of pigs themselves.

Introduction

Immunodeficient animal models are versatile tools in biomedical research. They are naturally used as research models of immunity. In addition, they can be used as animal platforms for the generation of humanized animals that steadily maintain transplanted tissues and/or cells of humans and can be used for in vivo biological research of human. They are also useful in the preclinical trials of stem cell-based regenerative medicine to evaluate the effectiveness and safety of the transplantation of human stem cells, such as iPS cells. Severely immunodeficient mice models, such as NOG (Ito et al., 2002), NSG (Shultz et al., 2005), and BRG (Traggiai et al., 2004) mice have been intensively developed. The functional reconstitution of the human hematopoietic and immune systems has been achieved by transplanting human hematopoietic stem cells into these immunodeficient mice (Ito et al., 2002, Shultz et al., 2005, Traggiai et al., 2004), and research on human-specific infections has been performed using these models (Akkina, 2013, Bility et al., 2012, Bility et al., 2014). These models have also been used in human cancer and stem cell research (Cunningham et al., 2012, Shultz et al., 2014). However, mice models have considerable limitations for modeling human biological and clinical conditions. Their small size narrows the scope of surgical and clinical applications, and their short longevity precludes long-term evaluation of post-transplantation effects. Moreover, there are several genetic lines of difference in adaptive and innate immune systems (Mestas and Hughes, 2004) as well as inflammatory responses (Seok et al., 2013) between mice and humans.

Pigs are invaluable animal models in human medical research because of their similarities to humans in physiology, anatomy, nutrition, and genetics. In fact, several genetically modified pigs have been shown to recapitulate human disease with higher fidelity than mice models (Fan and Lai, 2013, Prather et al., 2013). Therefore, immunodeficient pigs can be excellent animal models for biomedical research, such as the development of humanized tissues and organs for transplantation and long-term evaluation of transplanted cancer or stem cells of human origin. Our group (Suzuki et al., 2012) and Watanabe et al. (2013) previously reported the development of severe combined immunodeficient (SCID) pigs by disrupting the interleukin 2 receptor gamma (IL2RG) gene. These pigs had extremely hypoplastic thymi and expressed a T⁻B⁺NK⁻ phenotype. We also showed that these SCID pigs can be immune reconstituted by allogenic bone marrow (BM) transplantation (Suzuki et al., 2012). However, it is desirable that the SCID phenotype would be reinforced by additional gene modification(s) to achieve efficient engraftment of xenografts, including human stem cells.

Recombination activating gene (RAG) 1 and 2 are other genes where the inactivation of either gene induces a SCID phenotype in mice (Mombaerts et al., 1992, Shinkai et al., 1992), humans (Schwarz et al., 1996, Villa et al., 2001), and pigs (Huang et al., 2014, Ito et al., 2014, Lee et al., 2014). RAG1 and RAG2 collaborate in regulating the proper V(D)J rearrangements, which are indispensable for the generation of antigen receptor diversity in B cells and T cell receptor diversity in T cells (McBlane et al., 1995, Oettinger et al., 1990). The inactivation of RAG1 and/or RAG2 arrests lymphoid differentiation at an early stage and results in a lack of mature T and B cells. Therefore, we selected RAG2 as a target gene to be disrupted for the additional line in SCID pigs. Although the reports on RAG1- and/or RAG2-targeted pigs have already been published (Huang et al., 2014, Ito et al., 2014, Lee et al., 2014), they only showed the phenotypes of somatic-cell-cloned animals which can be accompanied with epigenetic abnormalities and they had not fully unraveled immunological traits of RAG-inactivated pigs. Therefore, additional research studies are still valuable in establishing the validated porcine SCID models. We report here the production of RAG2 knockout pigs via conventional gene targeting of somatic cells followed by somatic cell nuclear transfer and the characterization of immunological traits in their progenies which are free from epigenetic abnormalities. Moreover, we produced RAG2 and IL2RG double-knockout pigs toward the generation of more severe immunodeficient pigs which lacks not only T and B cells but also NK cells. The data presented here is useful for the development of an excellent humanized pig model as well as for veterinary research on porcine immunity.