Factors affecting the efficiency of somatic cell nuclear transplantation in the fish embryo.
Procedures to improve somatic cell nuclear transplantation in fish were evaluated. We reported effects of nonirradiated recipient eggs, inactivated recipient eggs, different combinations 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 gamma-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%).
Liu
Tong Ming
TM
State Key Laboratory of Freshwater Ecology and Biotechnology,Institute of Hydrobiology, The Chinese Academy of Sciences, Wuhan 430072, China.
Yu
Xiao Mu
XM
Ye
Yu Zhen
YZ
Zhou
Jian Feng
JF
Wang
Zhong Wei
ZW
Tong
Jing Gou
JG
Wu
Ching Jiang
CJ
D000818:Animals; D002347:Carps / Q000196:embryology* / Q000235:genetics; D002467:Cell Nucleus / Q000235:genetics; D002875:Chromosomes / Q000235:genetics; D019976:Cloning, Organism / Q000379:methods*; D016895:Culture Media, Serum-Free; D004625:Embryo, Nonmammalian / Q000166:cytology*; D047108:Embryonic Development; D005260:Female; D005720:Gamma Rays; D008297:Male; D018895:Microsatellite Repeats / Q000235:genetics; D053652:Nuclear Transfer Techniques; D010063:Ovum / Q000166:cytology / Q000502:physiology / Q000528:radiation effects
D016895:Culture Media, Serum-Free