Production of cloned rabbits by somatic nuclear transfer
Production of cloned rabbits by somatic nuclear transfer
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DOI：
10.1089/153623003772032808
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发表时间：
2003-12-01
期刊：
影响因子：
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通讯作者：
Renard, JP
中科院分区：
文献类型：
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作者：
Challah-Jacques, M;Chesne, P;Renard, JP
有两个技术因素似乎对兔克隆的成功至关重要：受体卵母细胞的激活时间（Collas 和 Robl，1990）以及受体子宫生理学与重建胚胎发育状态的同步（Chesne 等，2002）。在兔克隆方案中（Chesne 等，2002），每个供体细胞均与去核且未激活的中期 II 卵母细胞电融合。然后供体细胞的细胞核被转移到受体卵母细胞的细胞质中。由此产生的核移植 (NT) 胚胎放置 1 小时，以确保核膜破裂和染色体过早浓缩 (PCC)，因为成熟/中期促进因子 (MPF) 的活性在未激活卵母细胞的细胞质中仍然很高，(Campbell 等人，1996)。然后通过第二组电刺激激活 NT 胚胎，并在放线菌酮（一种蛋白质合成抑制剂）和 6-二甲氨基嘌呤（一种激酶抑制剂）存在下孵育，以降低 MPF 活性并允许进入 S 期。由于兔子受精卵在受精后很早就进入S期（Oprescu和Thibault，1965；Szöllösi，1966），因此这两种药物的暴露时间已缩短至1小时。处理卵母细胞时也应特别小心。兔卵母细胞很容易被激活。在体外，自发激活发生在排卵后 20-22 小时。温度的变化也会加速激活。迄今为止，只能使用新鲜收集的细胞来生产活体克隆兔（Chesne et al., 2002）。由于有兴趣使用兔子作为几种人类疾病（例如动脉粥样硬化、艾滋病、囊性纤维化等）的实验模型，我们正在积极致力于利用体细胞供体细胞生产活体动物，这些细胞经过多次传代培养，与实现靶向基因改造相一致。
TWO TECHNICAL FACTORS appear to be critical to the success of rabbit cloning: timing of activation of the recipient oocyte (Collas and Robl, 1990) and synchronization of the recipient uterus physiology with the development state of the reconstructed embryo (Chesne et al., 2002). In the rabbit cloning protocol (Chesne et al., 2002), each donor cell is electrofused with an enucleated and non-activated metaphase II oocyte. The nucleus of the donor cell is then transferred into the cytoplasm of the recipient oocyte. The resulting nuclear transfer (NT) embryos are left for 1 h to ensure nuclear envelope breakdown and premature chromosome condensation (PCC) because of the activity of maturation/metaphase promoting factor (MPF), which remains high in the cytoplasm of non-activated oocyte,(Campbell et al., 1996). The NT embryos are then activated through a second set of electrostimulation, and incubated in the presence of both cycloheximide (a protein synthesis inhibitor) and 6-dimethylaminopurine (a kinase inhibitor), to reduce the MPF activity and permit entrance into S phase. Because the rabbit zygote enters S phase very early after fertilization (Oprescu and Thibault, 1965; Szöllösi, 1966), the exposure period to these two drugs has been reduced to 1 h. Special care should also be taken in handling oocytes. Rabbit oocytes can be activated easily. In vitro, spontaneous activation occurs 20–22 h post ovulation. Variations in temperature can also accelerate activation.To date, producing live cloned rabbit has only been possible using freshly collected cells (Chesne et al., 2002). Because of the interests of using the rabbit as an experimental model for several human pathologies (eg, atherosclerosis, AIDS, cystic fibrosis,...), we are actively working to produce live animals from somatic donor cells cultured for a number of passages compatible with the realization of targeted genetic modification.