Generation of knockout rabbits with X-linked severe combined immunodeficiency (X-SCID) using CRISPR/Cas9

Yoshiko Hashikawa,Masaru Tajima,Ryuhei Hayashi,Tomoji Mashimo,Toru Okubo,Naofumi Takai,Yasuyuki Osada,Shohei Azuma,Kohji Nishida,Mitsuru Kuwamura,Yayoi Kunihiro

doi:10.1038/s41598-020-66780-6

Abstract

Severe immunodeficient mice are widely used to examine human and animal cells behaviour in vivo. However, mice are short-lived and small in size; while large animals require specific large-scale equipment. Rabbits are also commonly employed as experimental models and are larger than mice or rats, easy to handle, and suitable for long-term observational and pre-clinical studies. Herein, we sought to develop and maintain stable strains of rabbits with X-linked severe combined immunodeficiency (X-SCID) via the CRISPR/Cas9 system targeting Il2rg. Consequently, X-SCID rabbits presented immunodeficient phenotypes including the loss of T and B cells and hypoplasia of the thymus. Further, these rabbits exhibited a higher success rate with engraftments upon allogeneic transplantation of skin tissue than did wild type controls. X-SCID rabbits could be stably maintained for a minimum of four generations. These results indicate that X-SCID rabbits are effective animals for use in a non-rodent model of severe immunodeficiency.

Highlights

Immunodeficient animals are important for in vivo transplantation studies using human or animal cells
Large animals have a long period of sexual maturity and gestation period
We examined thymus tissue by HE-staining, indicating www.nature.com/scientificreports that thymus from X-linked severe combined immunodeficiency (X-SCID) rabbits contained almost no lymphocytes (Fig. 3d)

Summary

Introduction

Immunodeficient animals are important for in vivo transplantation studies using human or animal cells. Examples of X-SCID-animals include rats[7], marmosets[8], pigs[9], and dogs with the gene mutation[10] Functional defects such as T, B, and/or NK loss lead to severe immunodeficiency. Rabbits are the second most widely used animal after experimental rodent models owing to their long life, ease of handling, and the ability to be reared in a small space. They have a short period of sexual maturity and gestation and are more genetically related to humans than other rodents[11]. This study reports the development of Il2rg-KO rabbits and maintenance of a stable strain, using the CRISPR/Cas[9] system

Methods

Results

Conclusion