26 Production of Cloned Korean Raccoon (Nyctereutes Procyonoides Koreensis) Embryos by Interspecies Somatic Cell Nuclear Transfer Using Enucleated Pig Oocytes

Abstract

Interspecies somatic cell nuclear transfer (iSCNT) could be a useful method for embryo research of wildlife animals or endangered species. Because it is hard to obtain the oocytes or embryos of wildlife animals, its embryo research is not progressing well. Therefore, iSCNT is one of the alternative ways for wildlife animal embryo research and conservation of their genetic source. Until now, iSCNT has been applied to conservation of wildlife animals including guar, mouflon, banteng and African wildcat. The domestic pig oocytes have been used for iSCNT of other species such as tiger, sheep and dog and they successfully developed to the blastocyst stage. According to this concept, we performed wild-captured Korean raccoon (Nyctereutes procyonoides koreensis) iSCNT using porcine oocytes matured in vitro. Raccoon fibroblasts from ear skin samples of male raccoon were used as donor cells in 3 to 5 passages. The donor cells were cultured in DMEM supplemented with 15% FBS. Enucleated porcine oocytes were fused with raccoon fibroblasts by electrofusion. The iSCNT embryos were cultured in PZM-3 at 39°C for 7 days in an atmosphere of 5% CO2 and 5% O2. A total of 158 iSCNT embryos were cultured. More than 77% of the raccoon somatic cells successfully fused with the porcine oocytes; 68.5% of the iSCNT raccoon embryos developed to the 2- to 8-cell stage at Day 2 (1-cell: 9.7%, 2-cell: 14.4%, 4-cell: 34.1%, 6-cell: 12.7%, 8-cell: 7.3%, fragmented: 21.8%). This is similar to porcine SCNT results that 62.5% of the SCNT porcine embryos developed (1-cell: 8.0%, 2-cell: 4.2%, 4-cell: 23.6%, 6-cell: 13.6%, 8-cell: 23.8%, fragmented: 26.8%). But no embryos were further developed to blastocyst stage at Day 7 in iSCNT. In fragmentation evaluation in iSCNT embryos using by Hoechst stain at Day 2, two-cell stage embryos and four-cell stage embryos showed the normal numbers of nucleus. However, 6-cell stage embryos showed 4 to 5 nuclei and 8-cell stage embryos also showed 5 to 6 nuclei. Almost iSCNT embryos showed the developmental block at 4-cell stage embryos. This result was probably caused by an incomplete reprogrammed raccoon cell after iSCNT. Therefore, we treated with trichostatin A (TSA), a histone deacetylase inhibitor that has been used to enhance nuclear reprogramming following SCNT. Ninety-seven iSCNT raccoon-pig embryos were treated with 5 nM TSA during 15 h before being cultured in PZM-3. The TSA-treated iSCNT embryos showed similar developmental status to non-treated embryos (1-cell: 13.5%, 2-cell: 12.5%, 4-cell: 35.0%, 6-cell: 10.1%, 8-cell: 6.3%, fragmented: 22.5%). No embryos were further developed to blastocyst stage at day 7. Our results showed that 4-cell stage embryos of raccoon-porcine iSCNT embryos may be produced by iSCNT methods, but they were unable to support complete reprogramming of raccoon-porcine iSCNT embryos. This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. 007133022011), Rural Development Administration, Republic of Korea.