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The number of cells with the stemness property of HDDPC differs among individuals, which suggests that HDDPC showing an increased expression of both ALP and OCT-3/4 can be more easily reprogrammed to generate iPS cells after the forced expression of reprogramming factors.

The CRISPR/Cas9 system has enabled the editing of mammalian genomes; however, its applicability and efficiency in the pig genome has not been studied in depth. The α-gal epitope synthesized by α-1,3-galactosyltransferase gene (GGTA1) is known as a xenoantigen obtained upon pig-to-human xenotransplantation. We here employed the CRISPR/Cas9 system-mediated knock-in of endogenous GGTA1 via targeted homologous recombination (HR). Linearized donors with ~800-bp homology flanking the CRISPR/Cas9 target site [exon 4 (containing ATG) of GGTA1] served as a template for gene targeting by HR. Using a targeted toxin strategy to select clones lacking α-gal epitope expression, we successfully obtained several knock-in clones within 3 weeks of initial transfection. These results suggest that the use of CRISPR/Cas9-mediated HR to knock-in a mutated fragment at defined loci represents an efficient strategy to achieve the rapid modulation of genes of interest in swine cells and is a promising tool for the creation of KO piglets.

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