Nuclear Transplants from Adult Somatic Cells Generated by a Novel Method Using Diploidized Eggs as Recipients in Medaka Fish (Oryzias latipes)

Abstract

We previously reported the generation of fertile diploid adult fish with a donor marker by transfer of adult somatic cell nuclei to recipient diploidized eggs without enucleation in medaka (Oryzias latipes). Although transplants appeared similar to clones of donor fish, the possibility existed that they were chimeras of cells originating from both the donor and recipient nuclei. To clarify the nuclear origin of transplants, the green fluorescent protein gene (GFP) was used as the recipient marker and the DMY/dmrt1bY gene, which directs male differentiation in medaka, was used as the donor marker. The marker genes were examined in the transplants by fluorescence microscopy, polymerase chain reaction assays, and transmission to the progeny. Of the seven adult fish obtained from 974 nuclear transfer procedures, six were analyzed in detail. Three of these exhibited the donor phenotype but did not have the recipient marker, suggesting that they were donor clones. The other three showed GFP expression, with one exhibiting an apparent chimerism in both donor and recipient genetic markers and the other two considered to be parthenogenic. Elucidation of a mechanism capable of eliminating recipient nuclei from nuclear transplants is considered to be key to the establishment of cloning techniques in fish.