Characterization of Donor Cells for SCNT Mini-pig and Production of O Blood Type Knock-out Animals

Lim, J.S.; Lee, J.B.; Hur, C.G.; Sim, B.W.; Kim, M.J.; Cho, S.K.; Kang, K.S.

Characterization of Donor Cells for SCNT Mini-pig and Production of O Blood Type Knock-out Animals

2023

Somatic cell nuclear transfer (SCNT) in pig embryos constitutes an essential technique used to provide pharmacological proteins, xenotransplantation organs, and animal models for disease research. Following the first successful heart transplantation involving transgenic pigs, stabilization has emerged as the primary objective for subsequent investigations. The attainment of successful transformations fundamentally hinges upon the production of high quality SCNT embryos. Nonetheless, the efficiency of SCNT embryo production in pigs and mini-pigs remains low. Enhancing the efficiency of SCNT embryos is contingent upon several crucial factors, among which the type of donor cells assumes paramount importance. In this study, continuous subculture was used to produce donor cells to establish optimal conditions for the subculture of porcine fetal fibroblasts. Furthermore, it is imperative to comprehensively understand cell characteristics according to morphology. Moreover, to address the complications arising from blood type disparities, we selected the OO type for examination. The blood type of the mini pig was subjected to analysis and classification using PCR techniques. Following the OO type selection, sgRNA(single guide RNA) was employed in conjunction with CRISPR-Cpf1 to knock-out the GGTA1 (a-1,3 galactosyl-transferase) gene, thereby eliciting an acute immune response. A minipig cell line devoid of the Gal(1,3)Gal epitope was consequently constructed. This constructed cell line was then cloned through SCNT, with the resultant cloned embryo being transplanted into a surrogate mother to yield a GGTA1 knock-out mini pig. These findings serve as fundamental data for future research endeavors on xenotransplantation and SCNT.

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