Influence Of Different Enucleation Methods And Fusion,Activation Conditions On The Effect Of Mouse Somatic Cell Nuclear Transfer

In the study, the influence of different enucleation methods and fusion, activation conditions on the effect of mouse somatic cell nuclear transfer (SCNT) was studied, and the manipulation conditions were further optimized. The study was designed to pave the way for the application of the mouse somatic cell nuclear transfer.1. In the experiment, efficiency of oocytes enucleation with different holding methods, inner diameter of pipettes and oocytes obtained from different strain of mouse was studied. Results indicate that: (1) the improved enucleation method can reduce the mechanical damage to oocytes, and improve the survival rate of oocytes up to 82.13%, significantly higher (P<0.01)than that achieved by traditional method (23.60%); (2) There was no significant difference between the inner diamater of 10~12μm (63.13%) and 20μm (53.26%) pipettes but the surviving rate of enucleated oocytes significantly increased (P<0.05)when using the pipettes of 14~16μm inner diamater (80.87%); (3) The surviving rate of enucleated oocytes from strain of C57BL/6((?))×DBA((?))(B6D2F1) was significantly (P<0.01)higher (82.13%) than that from strain of ICR (49.77%).2. A single donor cell was injected into the perivitelline space of enucleated oocyte and fusion was induced with electric pulses. Results showed that (1) the fusion rate of donor cells in the media containing 0.32mol/L mannitol (52.28%) was significantly higher (P<0.01 )than that in the media containing 0.28mol/L and 0.36 mol/L mannitol(22.42% and 21.84%, respectively) ; (2) In the optimized fusion media, the fusion rate of tail-tip cells was 63.15% induced by a DC pulse of 800V/cm for 10μs; While using a DC pulse of 1000V/cm for 10μs, the fusion rate of fetus fibroblast cells was 67.68%, significantly higher (P<0.05) than other groups using different fusion parameter. (3) With the increase of the cell passage, the fusion rate of fetus fibroblast cells declined. The fusion rate of the primary and the first passage cells were 59.20% and 57.28% respectively, significantly higher (P<0.05) than that of the fifth passage cells.3. The experiment was conducted to study the effects on the activation of mouse oocytes under different concentration and duration of strontium chloride and different timing of activation. The results were as follows: (1) the highest activation percentage (86.61%) was obtained when the oocytes were treated with the calcium-free CZBG medium containing 5μg/mLCB and 4.0mmol/L SrCl2, significantly (P<0.05) higher than those obtained with the medium containing 2.0, 6.0, 8.0, 10.0 mmol/L SrCl2; (2)The activation rate (85.96%) achieved when the oocytes were treated with the calcium-free CZBG medium containing 5μg/mLCB and 4.0mmol/L SrCl2 for 180min was significantly (P<0.05) higher than those achieved with the same medium for 60min, 120min, 210min; (3) The activation rates of the oocytes collected 18 and 20 hours (82.07% and 85.44%) post hCG injection were significantly higher(P<0.05) than those recovered 14 and 16 hours (5.10% and 15.15%) post hCG under the same conditions.4. Preimplantation development of reconstructed embryos by nuclear transfer was studied. The reconstructed oocytes 389 were cultured in the calcium-free CZBG medium containing 5μg/mLCB and 4.0mmol/L SrCl2 for 3 hours, the activation rate was 53.49%. Parts of the reconstructed oocytes (80) were transferred into the oviducts of floater mother, after cultured for 72 hours in vivo, the embryos were recovered from uterus. The rates of reconstructed embryos from tail-tip cells and fetus fibroblast cells developed to 2-cell stage were 41.68% (5/12) and 27.94% (19/68), and to morula stage were 0% (0/12) and 5.88% (4/68) respectively.