Effect Of Different Enucleation Methods And Fusion,Culture Conditions On Mouse Somatic Cell Nuclear Transfer

The work was performed within a nuclear transfer program to investigate differentmethods for enucleating, fusing and culturing of reconstructed embryos. Fetal fibroblastcells were prepared from 13 dpc fetuses derived from B₆D₂F₁ embryos. B₆D₂F₁ strainwas also used for preparation of oocytes. During the culture of reconstructed embryos invitro, different development was observed by using different procedure of nucleartransfer.Experiment 1Double-holder was used for enucleation and nuclear transfer to increase the survivalrate of oocytes and minimize time-consumer of micromanipulation. Enucleation andinsertion of donor cell into the perivitelline space were accomplished sequentiallyduring a single round of manipulation. Double-holder is useful for a large size injectionpipette to manual penetrate the zona and remove the debris of the first polar body. Thesurvival rate of reconstructed oocytes was significantly higher by using the pipettes withinner diameter of 14～16μm(82.05%).Experiment 2The pre-treatment of PEG/DMSO on donor cells significantly improved thefusion rate (51.11% vs 25.64%).Effect of fusion midia containing different concentrations of mannitol and PVA onthe fusion rate of couplets was examined. Fusion rate in the fusion media containing0.1g/L PVA was higher than those in the media without PVA. Fusion rate in fusionmedia containing 0.32M mannitol (48.21%) was higher than those in the media with0.28M, 0.30M and 0.36M mannitol.Effect of different electrical field intensity on the fusion rate of couplets was alsostudied. The tip shape electrodes were used to fuse the couplets. Although the chamberis a much more routine way for fusing the reconstructed embryos, our study show that the tip shape electrodes can improve the fusion rates (42.31%, 43.13%), comparing toroutine parallel line electrodes (30.43%, 33.33%).Another test showed that the efficiency of electrofusion of oocytes with fetalfibroblast cells at found passage and the first passage was higher than at the second andthird passage (38.98%, 38.46%, 36.73% and 35.00%), regardless of other fusionconditions.Experiment 3Whether trichostatin A can improve the development of NT embryos was studied.We found that 5 nM TSA-treatment for 10 h following oocyte activation resulted inmore efficient in vitro development of somatic NT embryos.NT embryos cultured in KSOM medium showed a two-cell developmental rate of39.06%, which was higher than in CZBG and KSOM/Vero feed cell groups (34.14%,35.90%). However, two-cell stage NT embryos reach the morula stage at a higher rate(15.38%) in KSOM/Vero feed cell group.