Factors Affecting the Efficiency of Somatic Cell Nuclear Transplantation in the Fish Embryo TONG MING LIU, XIAO MU YU, YU ZHEN YE, JIAN FENG ZHOU, ZHONG WEI WANG, JING GOU TONG, and CHING JIANG WU n State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, The Chinese Academy of Sciences, Wuhan 430072, China ABSTRACT Procedures to improve somatic cell nuclear transplantation in fish were evaluated. We reported effects of nonirradiated recipient eggs, inactivated recipient eggs, different combina- tions between recipient eggs and donor cells, duration of serum starvation, generation number, and passage number of donor cells on developmental rates of nuclear transplant (NT) embryos. Exposure to 25,000 R of g-rays inactivated recipient eggs. Single nucleus of cultured, synchronized somatic cell from gynogenetic bighead carp (Aristichthys nobilis) was transplanted into nonirradiated or genetically inactivated unfertilized egg of gibel carp (Carassius auratus gibelio). There was no significant difference in developmental rate between nonirradiated and inactivated recipient eggs (27.27% vs. 25.71%, respectively). Chromosome count showed that 70.59% of NT embryos contained 48 chromosomes. It showed that most NT embryos came from donor nuclei of bighead carp, which was supported by microsatellite analysis of NT embryos. But 23.53% of NT embryos contained more than 48 chromosomes. It was presumed that those superfluous chromosomes came from nonirradiated recipient eggs. Besides, 5.88% of NT embryos were chimeras. Eggs of blunt-snout bream (Megalobrama amblycephala) and gibel carp were better recipient eggs than those of loach (Misgurnus anguillicaudatus) (25% and 18.03% vs. 8.43%). Among different duration of serum starvation, developmental rate of NT embryos from somatic nuclei of three-day serum starvation was the highest, reaching 25.71% compared to 14.14% (control), 20% (five-day), and 21.95% (seven- day). Cultured donor cells of less passage facilitated reprogramming of NT embryos than those of more passage. Recloning might improve the developmental rate of NT embryos from the differentiated donor nuclei. Developmental rate of fourth generation was the highest (54.83%) and the lowest for first generation (14.14%) compared to second generation (38.96%) and third generation (53.01%). J. Exp. Zool. 293:719–725, 2002. r 2002 Wiley-Liss, Inc. Cloning animals has become something of a cottage industry ever since Dolly first burst on the scene five years ago. Unlikely as it may seem, cloning attempts have met with little success. Numerous factors affect cloning efficiency of nuclear transplantation, including cell cycle stage of both donor nucleus and recipient cytoplasm, the differentiated state, genetic background, passage number of cultured cell, loss of imprints, accumu- lated genetic damage of the donor cell, or the ability of the oocyte to epigenetically reprogram the donor nucleus (Campbell, ’99; Rideout et al., 2000). In addition, recipient oocyte age, multiple pulse, manipulation medium, and cytochalasin B also affected the efficiency of nuclear transplanta- tion (Collas and Robl, ’90). In mammals, donor cells had been induced to exit the growth phase and caused changes in chromatin structure that facilitated reprogram- ming of gene expression and development of NT embryos by means of reducing the concentration of serum in the medium. Cells were arrested in G 0 /G 1 phase of the cell cycle by allowing them to grow to confluency followed by incubation for three days in medium containing 0.5% fetal calf serum (Mercer et al., ’90). Successful cloning of differentiated donor nuclei had been reported by reducing the concentration of serum in the medium from 10% to 0.5% for five days (Wilmut et al., ’97). Grant sponsor: National Natural Science Foundation of China; Grant number: 39830300. n Correspondence to: Ching Jiang Wu, State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, The Chinese Academy of Sciences, Wuhan 430072, China. E-mail: qjwu@ihb.ac.cn Received 30 October 2001; Accepted 11 July 2002 Published online in Wiley InterScience (www.interscience.wiley. com). DOI: 10.1002/jez.10177 r 2002 WILEY-LISS, INC. JOURNAL OF EXPERIMENTAL ZOOLOGY 293:719–725 (2002)