| Somatic cell nuclear transfer (SCNT) is a valuable method in farm animal improvement,drug production, regenerative medicine and conservation of invaluable genetic resources andespecially transgenic research in mammalian, establishing the SCNT transgenic model is animportant method in transgenic research and mouse has significant advantage as the model.However, the application of this technique is restricted by the low efficiency and highlyrequirement of skills in mouse. Micromanipulation methods with Piezo-impact unit assistedin mouse somatic cell nuclear transfer were optimized, and further, a complete mouse somaticcell nuclear transfer procedure was initially established. The study would be the basis forfurther research in establishing the SCNT transgenic mouse model. Following results wereobtained:1. Piezo-impact enucleation method was improved, nucleus was located by adjust theobject distance of Mâ…¡ oocytes in medium without sucrose, enucleation pipettes was cutthrough the zona pellucida using a few strong piezo pulses, aspirated the Mâ…¡ spindleandpulled out the oocytes. The efficiency of nuclear visualization and enucleation was studied.The result demonstrate the rate of visible nuclear was98.7%(153/155) by pretreatment of3%sucrose, compared98.3%(184/187) by adjust the object distance of Mâ…¡ oocytes inmedium without sucrose, there was no significant difference (P>0.05), but sucrosepretreatment increased exposure time and could result in wrinkle. The enucleation rate byimproved enucleation method in two micromanipulation mediums were84.6%,88.2%, andconventional method in two micromanipulation mediums were83.1%and88.6%respectively,still no significant differences among each other (P>0.05), suggested enucleation methodshave no significant effect on enucleation rate itself when the nuclear were visualized, butimproved method aspirated less cytoplasm accordingly to be thought better than conventionalmethod.2. Piezo-impact injection method was improved, method1was that enucleation pipettekeep negative pressure and aspirate a little cytoplasm while withdraw from the oocytes;method2was that enucleation pipette aspirate a little cytoplasm when withdrawed to theedge of the oocytes and gently pull out of the oocytes. The survival efficiency of enucleatedMâ…¡ oocytes by different injection methods was studied. The survival rate of enucleated Mâ…¡oocytes after injection with no sealing treatment was15.0%. The survival rate of enucleatedMâ…¡ oocytes by method1(43.9%) was significantly higher than that of controls (P<0.05), moreover, the survival rate of enucleated Mâ…¡ oocytes by method1(81.9%) was significantlyhigher than that by method1(P<0.05), suggested the two methods are both suitable forinjection, but method2is better for the recovery of the enucleated Mâ…¡ oocytes afterinjection.3. Mâ…¡ oocytes were activated by SrCl2; the activation efficiency of different activationmedium on Mâ…¡ oocytes was studied. The result showed that the activation rate anddevelopment rate of M16+SrCB were significantly higher than that of KSOM+SrCB(P<0.05), suggested M16+SrCB is more suitable for activation medium.4. The effect of different enucleation methods on the development of reconstructedembryos was then studied. Blastocysts were obtained only by improved enucleation methodin the micromanipulation medium without sucrose, and the development of8-cell (23.2%)and morula (23.2%) was also significantly higher than other groups (P<0.05), whichsuggested only improved enucleation method and micromanipulation medium withoutsucrose is better for the development of reconstructed embryos.5. The effect of different serum concentration in micromanipulation medium on thedevelopment of reconstructed embryos was also studied. Blastocysts were obtained only inthe medium with FBS, and the blastocysts rate of20%FBS group (10.9%) was significantlyhigher than10%FBS group (4.9%)(P<0.05). That suggested add20%FBS into themicromanipulation medium could enhance the development of reconstructed embryos.6. The effect of enucleation and injection on the nucleus and cytoskeleton of Mâ…¡oocytes. The results showed well-distributed microfilament in Mâ…¡ oocytes after enucleationand injection respectively, which had no significantly difference with that in Mâ…¡ oocyteswithout micromanipulation. But the distribution of microfilament in reconstructed embryosafter activation was irregular and not like in parthenogenetic. Suggested cytoskeleton ofoocytes were hurt by micromanipulation and it may be the reason for decrease ofdevelopment ability of reconstructed embryos.7.2-cell embryos obtained in vivo were transferred into the oviducts of pseudopregnantICR females, the pregnancy rate was33.3%, while no pregnancy evidence was seen afterreconstructed2-cell embryos transferred. |
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