Development of the Nude Rabbit Model

Abstract

Loss-of-function mutations in the forkhead box N1 (FOXN1) gene lead to nude severe combined immunodeficiency, a rare inherited syndrome characterized by athymia, severe T cell immunodeficiency, congenital alopecia, and nail dystrophy. We recently produced FOXN1 mutant nude rabbits (NuRabbits) by using CRISPR-Cas9. Here we report the establishment and maintenance of the NuRabbit colony. NuRabbits, like nude mice, are hairless, lack thymic development, and are immunodeficient. To demonstrate the functional applications of NuRabbits in biomedical research, we show that they can successfully serve as the recipient animals in xenotransplantation experiments using human induced pluripotent stem cells or tissue-engineered blood vessels. Our work presents the NuRabbit as a new member of the immunodeficient animal model family. The relatively large size and long lifespan of NuRabbits offer unique applications in regenerative medicine, cancer research, and the study of a variety of other human conditions, including immunodeficiency.

Keywords: FOXN1; immunodeficiency; nude rabbit; stem cell; xenotransplant.

Graphical abstract

Figure 1

Establishment of the NuRabbit colony (A) FOXN1 mutant indels that are present in the NuRabbit colony. Underlined in WT: Cas9 guide RNA targeting sequencing. (B) Breeding summary of NuRabbits.

Figure 2

Nude phenotype of NuRabbits (A–C) NuRabbits of different genotypes at 5 weeks of age. (A) Δ5/Δ10, (B) Δ5/Δ11, (C) Δ11/Δ11. (D) Representative photomicrographs of hair follicles in a Δ5/Δ11 NuRabbit (left) and a WT rabbit (right). Sections of skin in the NuRabbit (left) are characterized by dilated follicular ostia (arrowhead) containing variable amounts of keratin debris or irregularly formed hair shafts, accompanied by thinning of the follicular epithelium and decreased number and size of sebaceous glands (arrow) within follicular adnexa, compared with haired skin of WT rabbits (right), which have well-developed sebaceous units (arrow) and multiple follicles with intact hair shafts (arrowheads) per adnexa. Bars, 20 μm. (E) Hair patterns in Δ5/Δ10 NuRabbits at different ages. WT, wild type; HT, heterozygous FOXN1 knockout; Nu, NuRabbit.

Figure 3

NuRabbits are immunodeficient (A) Lack of thymus development in a NuRabbit (right), compared with normal thymus development in a WT rabbit (circled in blue). (B) Sections of thymus (bar, 100 μm), spleen (bar, 50 μm), and appendix (bar, 20 μm) from WT rabbits (left) compared with NuRabbits (right). Compared with thick cortical lymphoid areas (arrowheads) in the thymus of WT rabbits, NuRabbits show marked reduction in lymphoid elements, with only a few clusters of cells remaining in loose connective tissue stroma of thymic lobules. The spleen of WT rabbits has large lymphoid follicles (arrowheads), compared with a general paucity of lymphoid tissue in NuRabbits, with only small remnants remaining (arrowhead). In sections of the appendix, WT rabbits have dense sheets of lymphoid tissue within the lamina propria, compared with a patchy loss of lymphocytes in NuRabbits, accompanied by pyknotic cellular debris (arrowhead) and large foamy macrophages phagocytosing degenerate lymphocytes (arrow). (C) Representative flow cytometry results of peripheral blood lymphocytes in WT and NuRabbits. (D) Summary of B and T cell populations in peripheral blood from seven NuRabbits in comparison with that from three WT rabbits.

Figure 4

NuRabbits support iPSC-derived teratoma growth (A) Illustration of teratoma test in NuRabbits. (B) Summary of teratoma formation in NuRabbits. (C) Representative pictures of teratomas generated in NuRabbits and NuMice. (D) Germ-layer staining of teratomas obtained from NuMice and NuRabbits. Arrowheads point to endoderm-derived epithelium (D1 and D4), mesoderm-derived nervous system tissues (D2 and D5), and ectoderm-derived cartilage foci (D3 and D6). Scale bars, 20 μm in D1; 50 μm in D2, D3, and D4; 20 μm in D5; and 50 μm in D6. (E) Summary of teratoma sizes in NuRabbits and NuMice.

Figure 5

Transplantation of TEBV to NuRabbit (A) Illustration of patient-specific stem cell-derived TEBVs. (B) Representative images of common carotid arteries from an adult mouse (left) and an adult rabbit (right). D, diameter. (C) A representative TEBV. (D) Representative image of a TEBV after transplantation to the left common carotid artery in a NuRabbit.