Parthenogenesis is maternally uniparental reproduction through the embryonic development of oocytes without fertilization. Artificial activation of mature oocytes could generate homozygous haploid embryos with the extrusion of the second polar body. However, the haploid embryos showed low embryo development in preimplantation embryos. In this study, we investigated whether the electronic fusion of the haploid embryos could enhance embryo development and ESC establishment in mice. Haploid embryos showed the developmental delay from 4-cell to the blastocyst stage. The haploid blastomeres of the 2-cell stage were fused electronically, resulting in that the fused embryos showed a significantly higher rate of blastocysts compared to non-fused haploid embryos (55% vs. 37%). Further, the embryonic stem cells (ESCs) derived from the fused embryos were confirmed to be diploid. The rate of ESC establishment in fused embryos was significantly higher compared to non-fused ones. Based on the results, we concluded that the electronic fusion of haploid embryos could be efficient to generate homozygous ESCs.

Transplantation is considered to be a very useful approach to improve human welfare and to prolong life-span. Heterologous organ transplantation using pig organs which are similar to human beings and easy to make mass-production has known as one of the alternatives. To ensure potential usage of the pig organ for transplantation application, it is essentially required to generate transgenic pig modifying immuno-related genes. Previously, we reported production of heterozygous α 1,3-galactosyltransferase (GalT) knock-out and human membrane cofactor protein (MCP) expressing pig (GalT-MCP/+), which is enforced for suppression of hyperacute and acute immunological rejection. In this study, we reported generation of homozygous pig (GalT-MCP/-MCP) by crossbreeding GalT-MCP/+ pigs. Two female founders gave birth to six of GalT-MCP/-MCP, and seven GalT-MCP/+ pigs. We performed quantitative real-time PCR, western blot, and flow cytometry analyses to confirm GalT and MCP expression. We showed that fibroblasts of the GalT-MCP/-MCP pig do not express GalT and its product Gal antigen, while efficiently express MCP. We also showed no expression of GalT, otherwise expression of MCP at heart, kidney, liver and pancreas of transgenic pig. Taken together, we suggest that the GalT-MCP/-MCP pig is a useful candidate to apply xenotransplantation study.

Even though klotho deficiency in mice exhibits multiple aging-like phenotypes, studies using large animal models such as pigs, which have many similarities to humans, have been limited due to the absence of cell lines or animal models. The objective of this study was to generate homozygous klotho knockout porcine cell lines and cloned embryos. A CRISPR sgRNA specific for the klotho gene was designed and sgRNA (targeting exon 3 of klotho) and Cas9 RNPs were transfected into porcine fibroblasts. The transfected fibroblasts were then used for single cell colony formation and 9 single cell–derived colonies were established. In a T7 endonuclease I mutation assay, 5 colonies (#3, #4, #5, #7 and #9) were confirmed as mutated. These 5 colonies were subsequently analyzed by deep sequencing for determination of homozygous mutated colonies and 4 (#3, #4, #5 and #9) from 5 colonies contained homozygous modifications. Somatic cell nuclear transfer was performed to generate homozygous klotho knockout cloned embryos by using one homozygous mutation colony (#9); the cleavage and blastocyst formation rates were 72.0% and 8.3%, respectively. Two cloned embryos derived from a homozygous klotho knockout cell line (#9) were subjected to deep sequencing and they showed the same mutation pattern as the donor cell line. In conclusion, we produced homozygous klotho knockout porcine embryos cloned from genome-edited porcine fibroblasts.

To overcome the hyperacute immune rejection during pig-to-non-human primates xenotranasplantation, we have produced and bred α-1,3-galactosyltransferase knock-out (GalT —/—) pigs. In this study, the somatic cells and tissues from the GalT —/— pigs were characterized by an analysis of the expression of Galα-1,3-Gal (α-Gal) epitope. Briefly, ear fibroblast cell lines of 19 homozygous GalT —/— pigs were established and cryopreserved. The expression of α-Gal epitope in the cells was measured by fluorescence activated cell sorter (FACS) analysis using BS-I-B4 lectin. Also, the homozygous (GalT —/—) cells and tissues samples were immunostained with BS-I-B4 lectin for analysis of α-Gal epitope expression. The results showed that the expression of α-Gal epitope in GalT —/— cells (0.2 %) were significantly (p< 0.05) down-regulated to the range of cynomolgus monkey fibroblast (0.2 %) cells compared to heterozygous (GalT —/+) (9.3 %) and wild type (GalT +/+) (93.7 %) fibroblast cells. In the immunostaining results, while the expression of α-Gal epitope was detected a partly in GalT —/+ cells and mostly in GalT +/+ cells, it was almost not detected in the GalT —/— cells. Also, immunostaining results from various tissues of the GalT —/— pig showed that the expression of α-Gal epitope was not detectable, whereas various tissues from GalT +/+ pig showed a strong expression of α-Gal epitope. Our results demonstrated that α-Gal epitope expressions from GalT —/— pigs were successfully knocked out to prevent hyperacute immune rejection for further study of xenotransplantation.