Earticle

For the pig to human organ transplantation, immune rejection is the biggest problem. It is known that T cell is associated with immune rejection and it is necessary to produce severe combined immune deficiency (SCID) pigs to reduce the rejection. Previous studies show that IL2RG and RAG2 mainly function on T cell development which is mainly functions on immune system. Especially, IL2RG is for the T cell and NK cell development, and RAG2 is for maturation of T and B cells. In this study, to produce IL2RG and RAG2 deficient cell lines, we designed specific sgRNAs which target pig IL2RG and RAG2, respectively, using CRISPR/Cas9 system. Each of the GFP tagged targeting vectors were constructed and transfected into pig fibroblasts, respectively or both, to establish single or double knock-out cell lines. After transfection, GFP positive cells were isolated and many single cells were freely seeded and cultured individually. To analysis genomic types of the cells, we amplified and analyzed the targeting regions on DNA. As a result, 3 knock-out cell lines were established for IL2RG and RAG2, respectively. Also, for the double knock-out cell lines, 1 cell line was analyzed by sequencing. By CRISPR/Cas9 system, we established diverse IL2RG and/or RAG2 knock-out cell lines and the cells are planning on producing SCID pigs.