Improved efficiency of bovine somatic cell nuclear transfer by optimizing operational procedures.
To improve bovine somatic cell nuclear transfer (SCNT) efficiency, we studied various aspects to optimize the experimental procedures. Firstly, donor cells were treated with pronase, which resulted in a higher fusion rate than that of cells without the pronase treatment (78.3 vs. 53.9%). Secondly, when fused embryos were activated either by chemical (ionomycin + cyclohemixide (CHX)) or electrical + CHX stimulation, the cleavage and blastocyst formation rates were comparable amongst these treatment groups (P>0.05); however, mortality following electrical + CHX activation was significantly higher than that observed with the chemical activation, regardless of the pronase treatment (P<0.05). Finally, we compared the culture conditions of the reconstructed embryos using ACM medium plus mouse embryonic fibroblasts (MEF) vs. B2 medium plus granulose cells (GC), and the results clearly demonstrated that the former culture conditions led to a higher blastocyst rate, 90-day pregnancy rate, and newborn rate, than that observed for culture in B2 medium plus GC (46.7 vs. 34.7%, 36.1 vs. 9.6% and 25.9 vs. 5.8% for the blastocyst, pregnancy and newborn rates, respectively). In summary, the efficiency of bovine SCNT can be greatly improved using optimized operational procedures, including treating the donor cells with pronase, activation of fused embryos by ionomycin + CHX and the culture of the reconstructed embryos in ACM + MEF media.
Zhao
Lei-Wen
LW
Institute of Medical Genetics, Shanghai Jiaotong University, Shanghai, China.
Yang
Xiao-Yu
XY
Guan
Peng-Fei
PF
Fu
Jing
J
Li
Hua
H
Zhou
Yi-Ye
YY
Huang
Shu-Zhen
SZ
Zeng
Yi-Tao
YT
Zeng
Fan-Yi
FY
D000818:Animals; D002417:Cattle / Q000502:physiology*; D003513:Cycloheximide / Q000494:pharmacology; D004558:Electric Stimulation; D046149:Embryo Culture Techniques / Q000662:veterinary*; D005260:Female; D005347:Fibroblasts / Q000166:cytology; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D053652:Nuclear Transfer Techniques / Q000662:veterinary*; D009865:Oocytes / Q000166:cytology; D011247:Pregnancy; D018873:Pregnancy Rate; D011402:Pronase / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012098:Reproduction / Q000502:physiology*
D007476:Ionophores; D011500:Protein Synthesis Inhibitors; D015759:Ionomycin; D003513:Cycloheximide; D011402:Pronase