iSCNT embryo culture system for restoration of Cervus nippon hortulorum, presumed to be sika deer in the Korean Peninsula

Abstract

Sika deer inhabiting South Korea became extinct when the last individual was captured on Jeju Island in Korea in 1920 owing to the Japanese seawater relief business, but it is believed that the same subspecies (Cervus nippon hortulorum) inhabits North Korea and the Russian Primorskaya state. In our study, mt-DNA was used to analyze the genetic resources of sika deer in the vicinity of the Korean Peninsula to restore the extinct species of continental deer on the Korean Peninsula. In addition, iSCNT was performed using cells to analyze the potential for restoration of extinct species. The somatic cells of sika deer came from tissues of individuals presumed to be Korean Peninsula sika deer inhabiting the neighboring areas of the Primorskaya state and North Korea. After sequencing 5 deer samples through mt-DNA isolation and PCR, BLAST analysis showed high matching rates for Cervus nippon hortulorum. This shows that the sika deer found near the Russian Primorsky Territory, inhabiting the region adjacent to the Korean Peninsula, can be classified as a subspecies of Cervus nippon hortulorum. The method for producing cloned embryos for species restoration confirmed that iSCNT-embryos developed smoothly when using porcine oocytes. In addition, the stimulation of endometrial cells and progesterone in the IVC system expanded the blastocyst cavity and enabled stable development of energy metabolism and morphological changes in the blastocyst. Our results confirmed that the individual presumed to be a continental deer in the Korean Peninsula had the same genotype as Cervus nippon hortulorum, and securing the individual's cell-line could restore the species through replication and produce a stable iSCNT embryo.

Fig 1. Deer tissue culture and SCNT process.

The photographs above show the successive stages of culturing deer’s ear tissue to produce a cell-line. The photographs below show generation of SCNT-derived embryos and their subsequent in vitro culture. (Scale bar  =  100 μm).

Fig 2. Phylogenetic analysis of mt-DNA isolated from sika deer based on the neighbor-joining method by MEGA 6.0.

Fig 3. Development of blastocysts in each culture condition.

A Development of SCNT embryos in each culture medium. B The cavity analysis is a photo edited in reverse after phase difference microscopy. The gray shaded photograph was taken by enlarging only one blastocyst. The red arrow points to the blastocyst cavity. The white arrow points to the trophectoderm.

Fig 4. Development of SCNT-derived embryos in NCSU-23 medium, supplemented A or not supplemented B with hormones (P4 hormones), in co-culture with endometrial cells.

Small picture: Hoechst 33258 staining.