Reproduction Abstracts

Pigs are an excellent model for human diseases with their physiological similarities to human. In addition, they are assumed to be the most suitable organ donor to meet acute shortage of allogenic human organs. However, prior to their use in clinical studies, several genetic modifications are required to generate human diseases and to overcome immunological rejection of the xenograft, and specific breeding scheme of them is needed to avoid the transmission of zoonotic diseases. While the former can be addressed by introduction and/or knock-out (KO) of relevant genes by somatic cell nuclear transfer (SCNT), the later can be achieved by producing and breeding gnotobiotic pigs. Recently, we have been successful in generating and breeding the MHC class II homozygous pigs in a barrier-sustained, gnotobiotic miniature pig facility. Here, we describe a rapid single-step procedure to produce the specific gene KO pigs for xenotransplantation. We have been established a suitable transfection and selection procedures to produce efficiently KO somatic cells and utilized them as donor cells in producing KO pigs with SCNT technology. First, we produced CMP-N-acetylneuraminic acid hydroxylase (CMAH) KO pigs by this rapid single-step ZFN procedure. Second, we generated the SCID pigs with KO of RAG2 gene. Interestingly, these KO pigs supported the formation of teratomas in the injection sites of human stem cells. Therefore, our gnotobiotic pig facility and transgenic pigs may be used as a valuable resource for xenotransplantation and human diseases.