Generation of Fertile and Diploid Fish, Medaka (Oryzias latipes), from Nuclear Transplantation of Blastula and Four-Somite-Stage Embryonic Cells into Nonenucleated Unfertilized Eggs

Abstract

In two experimental series of transplantation of embryonic cell nuclei into nonenucleated unfertilized eggs in medaka (Oryzias latipes), fertile and diploid nuclear transplants were successfully generated. In the first experiment, nuclei from blastula cells of a medaka stock with the wild-type body color were transplanted into 1722 eggs from the orange-red variety. Of 26 adult nuclear transplants with the wild-type body color, 22 were, as expected, triploid and sterile, but the other four were fertile. Three of the four were diploid, and the last one was tetraploid. They transmitted the wild-type body color to the F1 and F2 progenies in a Mendelian fashion. In the second experiment, cell nuclei from four-somite-stage embryos of the orangered variety carrying the green fluorescent protein (GFP) transgene were transplanted into 1688 recipients of the same strain. Three adult nuclear transplants expressing GFP were obtained. Two of them were triploid and sterile, but the remaining one was fertile and diploid. The transgene of the donor nuclei was transmitted to the F(1) and F(2) offspring in a Mendelian fashion. These observations that diploid and fertile nuclear transplants could be obtained without enucleation of the recipient eggs may have important implications for future nuclear transplantation in medaka.

Diploidized eggs reprogram adult somatic cell nuclei to pluripotency in nuclear transfer in medaka fish (Oryzias latipes)

“…We also observed, in rare cases, the generation of diploid and fertile adults by transfer of nuclei from early or later embryonic cells into non-enucleated recipient eggs, though, in most cases, triploid and infertile adults were the result, as expected (Bubenshchikova et al 2005). However, when we used adult cell nuclei as donors in the transfer to enucleated recipient eggs, the development of transplant embryos was considerably weak (Wakamatsu, unpubl.…”

“…1B) (Bubenshchikova et al 2005). In this strain, GFP expression is first visually detectable (weakly) in the premid-gastrula stage (stage 14), and is clearly visible throughout the embryo after the two-somite stage (stage 19).…”

“…Cells that had migrated from the tissue fragments, most of which expressed GFP (Fig. 1C), were trypsinized and stored in L-15 for up to 5 h at 4°C until used (Bubenshchikova et al 2005). More than 95% of these cells were diploid, and the remaining less than 5% were tetraploid.…”

Ploidy mosaicism in well‐developed nuclear transplants produced by transfer of adult somatic cell nuclei to nonenucleated eggs of medaka (Oryzias latipes)

“…In contrast to the first cell cycle, synchrony between the cell cycles of the donor nuclei and the cytoplasm in the transplant embryos occurs after the first cytokinesis, resulting in well-developed transplants expressing the donor marker gene. The formation of triploid nuclei by fusion of a haploid nucleus originating from the recipient nucleus with the donor diploid nucleus in the same blastomere at the two-cell stage in the transplant embryos is similar to the generation of healthy but infertile triploid transplants from nuclear transfer using early embryonic cell nuclei as donors and nonenucleated unfertilized eggs as recipients (Niwa et al 1999;Bubenshchikova et al 2005). In this transfer, the triploid transplants were thought to be generated by fusion of the donor pluripotent diploid nuclei with the recipient haploid nuclei in the cytoplasm of transplant embryos during the first cell cycle.…”

“…At 4-6 days after inoculation, cells that had migrated from the tissue fragments, most of which expressed GFP (Fig. 1D), were trypsinized and stored in L-15 for up to 5 h at 4°C until use (Bubenshchikova et al 2005). More than 95% of the cells contained normal diploid chromosome sets (48 chromosomes) while the remaining less than 5% were tetraploid (96 chromosomes).…”