Biallelic modification of IL2RG leads to severe combined immunodeficiency in pigs.

Jung-Taek Kang,Nathaniel Jue,Jinseok Lee,Yun-Jin Yun,Kiho Lee,Eun-Jin Lee,Dal-Young Ji,Kwang-Wook Park,Sherrie Clark-Deener,Sunmi Ahn,Caitlin Ray,Junghyun Ryu,Bumrae Cho

doi:10.1186/s12958-016-0206-5

Abstract

sBackgroundPigs with SCID can be a useful model in regenerative medicine, xenotransplantation, and cancer cell transplantation studies. Utilizing genome editing technologies such as CRISPR/Cas9 system allows us to generate genetically engineered pigs at a higher efficiency. In this study, we report generation and phenotypic characterization of IL2RG knockout female pigs produced through combination of CRISPR/Cas9 system and SCNT. As expected, pigs lacking IL2RG presented SCID phenotype.MethodsFirst, specific CRISPR/Cas9 systems targeting IL2RG were introduced into developing pig embryos then the embryos were transferred into surrogates. A total of six fetuses were obtained from the embryo transfer and fetal fibroblast cell lines were established. Then IL2RG knockout female cells carrying biallelic genetic modification were used as donor cells for SCNT, followed by embryo transfer.ResultsThree live cloned female piglets carrying biallelic mutations in IL2RG were produced. All cloned piglets completely lacked thymus and they had a significantly reduced level of mature T, B and NK cells in their blood and spleen.ConclusionsHere, we generated IL2RG knockout female pigs showing phenotypic characterization of SCID. This IL2RG knockout female pigs will be a promising model for biomedical and translational research.Electronic supplementary materialThe online version of this article (doi:10.1186/s12958-016-0206-5) contains supplementary material, which is available to authorized users.

Highlights

Pigs with Severe Combined Immunodeficiency (SCID) can be a useful model in regenerative medicine, xenotransplantation, and cancer cell transplantation studies
Transferring in vitro fertilization (IVF) embryos injected with the RNA form of CRISPR/Cas9 system resulted in one pregnancy (Table 2) and six fetuses were collected from the surrogate to establish IL2RG knockout cell lines
The fetuses and cell lines were screened by the T7 endonuclease I (T7E1) assay and PCR DNA sequencing to detect potential mutation generated by the CRISPR/Cas9 system

Summary

Introduction

Pigs with SCID can be a useful model in regenerative medicine, xenotransplantation, and cancer cell transplantation studies. We report generation and phenotypic characterization of IL2RG knockout female pigs produced through combination of CRISPR/Cas system and SCNT. The receptor is a key part of major lymphocytes, supports the growth and maturation of several subtypes of lymphocytes: T cells, B cells, and natural killer (NK) cells. These lymphocytes are an essential component of the adaptive and innate immune system. Deletion or mutation in the IL2RG gene would lead to the loss of functional immune system.

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Discussion

Conclusion