化学去核卵母细胞为受体的小鼠体细胞核移植  

Reconstruction of mouse embryos with chemically enucleated oocytes

作　　者：杜卫华[1] 李世杰[2] 郑敏[1] 戴蕴平[1] 李宁[1] 

机构地区：[1]中国农业大学农业生物技术国家重点实验室,北京100094 [2]中国农科院畜牧所胚胎工程与繁殖技术室

出　　处：《广西农业生物科学》2006年第B09期201-202,共2页Journal of Guangxi Agricultural and Biological Science

基　　金：国家863计划项目(2001AA213091);北京市自然科学基金重大项目(5030001)

摘　　要：卵母细胞的化学去核是采用干扰染色体分离或纺锤体正常功能的化学试剂,使其所有染色质通过纺锤丝牢固结合,并借助极体排出的惯性将所有染色质全部带出胞外,达到去核目的。目前以化学去核处理的MⅡ期卵母细胞为受体,已获得克隆小鼠。然而,第一次减数分裂期的小鼠卵母细胞经化学去核后,进行核移植还未见报道。与传统的机械去核相比,该方法对卵母细胞无机械损伤,完全是极体的自然排放;同时细胞质及其中核重编程相关因子损失量小;而且高效、省时,程序简单,所得的卵胞质或许更适合于供体细胞核的重编程。剪取超排小鼠的卵巢,以注射器刺破有腔卵泡后获得卵母细胞和卵丘细胞复合体,进行体外成熟培养。成熟培养5 h后去除卵丘细胞,挑选生发泡破裂的细胞顺序移入含脱羰秋水仙碱(DC,0.4μg/mL,2 h)和DC(0.4μg/L)+放线菌酮(CHX,50μg/mL)的M16培养液中继续培养,直到第一极体排出。去核卵胞质与胎儿成纤维细胞用植物凝集素(PHA,200μg/mL)粘合后,转入电击槽;施加1个5 V/mm、3μs交流电脉冲和2个92 V/mm、70μs直流电脉冲进行电融合。3 h后,以SrCl2激活6h,于四孔培养皿中制作的“孔中孔”(well of well)体外培养重构胚。试验重复3次,共计698个卵母细胞,获得的重构胚融合率和激活率分别为84.8%和93.6%;胚胎2-细胞发育率为24.7%,4-细胞率为6.74%;2-细胞期克隆胚移植假孕受体后,没有获得怀孕受体。试验分别以“血清饥饿”胎儿成纤维细胞、新鲜细胞和冷冻保存细胞为供体作核移植,结果表明,冷冻保存细胞的融合率(69.3%)与其余两组(80.6%和84.8%)呈显著差异(P<0.05);激活率、2-细胞和4-细胞发育率,则三组间差异不显著(P>0.05)。本研究将化学去核与无透明带技术相结合,完全丢弃了传统核移植的显微操作及其繁琐程序,属手工克隆,它的成功将会大大简化核移植程序,同时提高了Induced enucleation is carried out with agents disturbing the meiosis or inhibiting protein synthesis of oocyte during polar body emites. Consequently all chromatin are extruded as the form of polar body. The mice have been cloned from embryonic stem cell karyoplasts and activated cytoplasts prepared by induced enucleation of MⅡ oocytes. However the nuclear transfer of mice with the induced-enucleation M Ⅱ oocytcs as the recipient has not been reported. Compared to the traditionally micromanipulation, the induced enucleation with chemical does not damage the cytoplast cytoskeleton, reduce the volume and cause the lost of some important factors involved in reprogramming of donor karyoplast. Moreover its high efficiency, time-saving and simple procedures maybe make the cytoplast derived from the induced enucleation more suitable to the reprogramming of donor nuclear. Ovaries were removed from animals and immersed in M2. The GV oocytes were collected by puncturing the large antral follicles using 25-gauge needles and matured in vitro. After 5 h IVM, oocytes that had undergone GVBD were cultured in M16 containing DC for 2 h, then in M16 with DC and CHX sequently until the first polar body was extruded. Completely enucleated oocytes were transferred into the M2 medium with 0.5% protease to remove the zona. Then the mouse fetal fibroblast cells were glued to the oocytes membrane with PHA. Consequently the oocyte-cell couplets were fused by electronic pulse and the fused couplets were transfer into medium with SrCla for 6 h. Finally the reconstructed embryos were cuhured in well of well with M16. In total, 698 oocytes were treated in our experiment with three repeats. The rates of fused and activated reconstruction embryos were 84.8G and 93.6G respetively. Embryos in 4-cell stage were derived by this method. No pregnancy was observed after the 2-cell embryos were transferred to the surrogate mice. Additionally, the effects of 3 protocols for donor preparation （trypsinizing the cultured cells with or without

关 键 词：化学去核 小鼠 无透明带技术 体细胞核移植 

分 类 号：S813[农业科学—畜牧学]