猪卵丘细胞核移植研究

Study on Cumulus Cell Nuclear Transfer in Porcine

以卵丘细胞为供核体细胞,采用胞质内注射法进行猪体细胞核移植,对去核、激活和培养等关键技术过程进行研究,结果表明,①点压法、挤压法对卵母细胞去核率明显高于盲吸法(三者分别为62.5%,64.6%,50.7%,P<0.05)。盲吸法去核染色体容易发生位置偏离,影响去核效果,但对早成熟的卵母细胞(36h～44h)进行去核可明显提高去核效率(P<0.05),在成熟培养36h～38h、39h～41h、42h～44h去核率分别为60.9%、67.8%、64.3%,而45h～48h为48.4%。②体细胞预激活有助于提高核移胚卵裂率(28.0%,20.1%,P<0.05)。A23187(Ca2+ionophore,钙离子载体)单独或与6DMAP(6Dimethylaminopurine,6二甲基氨基嘌呤)联合作用能使猪体细胞核移胚激活继续发育。③核移胚以胚胎培养液NCSU23(NorthCarolinaStateUniversity23medium,北卡洲立大学培养液23)及卵丘颗粒单层共培养体系进行分别培养,核移胚卵裂率无明显差异(30.06%,31.5%,P>0.05)。但NCSU23培养4细胞后发育能力更高(13.5%,3.9%,P<0.05)。

Some key effect factors such as enucleation,activation and culture in the course of porcine somatic cells nuclear transfer(NT) using cumulus donor nuclei were examined,in the method of microjection into cytoplasm of enucleated oocytes.The results were as follows:First,point-pressing method(PPM) and extruding mathod(EM) were significantly higher than blind aspiration method(BAM) in enucleation rate (62.5%/64.6%:50.7%,P<0.05).Enucleation efficiency was affected with chromosomes deviated in BAM.But the rate of enucleation could be promoted when oocytes were enuleated in early maturation stage (36-38 h 60.9%;39-41 h,67.8%,41-44 h,64.3%,respectively), significantly higher than in 44-48 h(48.4%).Second,when somatic cells were preactivated,the development rate of NT embryos increased significantly,increased from 20.1% to 28.0%.A23187 can lead NT embryos to active and develop when used respectively or combined with 6-DMAP. Third,when NT embryos were cultured in NCSU-23 medium(North Carolina State University-23 medium) ,the development rate(30.06%) was no significantly different from that in TCM199 containing cumulus single layer culture system(31.5%).However,the rate of development to 4-cell stage or above 4-cell cultured in NCSU-23 of NT embryos was increased(13.5%),which was significantly different in TCM199- cumulus system(3.9%).