Reprogramming following somatic cell nuclear transfer in primates is dependent upon nuclear remodeling
Reprogramming following somatic cell nuclear transfer in primates is dependent upon nuclear remodeling
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DOI：
10.1093/humrep/dem136
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发表时间：
2007-08-01
影响因子：
6.1
通讯作者：
Wolf, D. P.
中科院分区：
文献类型：
--
作者：
Mitalipov, S. M.;Zhou, Q.;Wolf, D. P.
背景：体细胞核移植（SCNT）需要细胞质介导的供体核重编程。成熟促进因子等细胞质因子参与核膜破裂（NEBD）和染色体过早凝聚（PCC）。鉴于以往在灵长类动物中使用传统方法进行SCNT的困难，我们假设细胞质诱导核重塑的能力有助于有效的重编程。方法：采用层粘连蛋白A/C免疫标记法检测猴SCNT胚胎NEBD和PCC。结果：最初，观察到供体细胞核与细胞质融合后持续的层粘连蛋白a /C信号，表明SCNT后NEBD不完整，并预测发育停止。然后，我们确定了荧光染料辅助去核和供体细胞电融合可能是诱导细胞质过早激活和随后缺乏核重塑的候选者。设计用于防止SCNT期间细胞质过早激活的改进方案显示出强大的NEBD和PCC。巧合的是，超过20%用胎儿成纤维细胞重建的SCNT胚胎进展为囊胚。在其他体细胞中也得到了类似的结果。重建囊胚显示Oct-4表达模式与受精胚胎相似，反映了成功的重编程。结论：我们的研究结果在阐明核重塑事件在SCNT后重编程中的作用方面取得了重大突破。
BACKGROUND: Somatic cell nuclear transfer (SCNT) requires cytoplast-mediated reprogramming of the donor nucleus. Cytoplast factors such as maturation promoting factor are implicated based on their involvement in nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC). Given prior difficulties in SCNT in primates using conventional protocols, we hypothesized that the ability of cytoplasts to induce nuclear remodeling was instrumental in efficient reprogramming.METHODS: NEBD and PCC in monkey (Macaca mulatta) SCNT embryos were monitored by lamin A/C immunolabeling.RESULTS: Initially, a persistent lamin A/C signal from donor cell nuclei after fusion with cytoplasts was observed indicative of incomplete NEBD following SCNT and predictive of developmental arrest. We then identified fluorochrome-assisted enucleation and donor cell ellectrofusion as likely candidates for inducing premature cytoplast activation and a consequent lack of nuclear remodeling. Modified protocols designed to prevent premature cytoplast activation during SCNT showed robust NEBD and PCC. Coincidently, over 20% of SCNT embryos reconstructed with fetal fibroblasts progressed to blastocysts. Similar results were obtained with other somatic cells. Reconstructed blastocysts displayed patterns of Oct-4 expression similar to fertilized embryos reflecting successful reprogramming.CONCLUSIONS: Our results represent a significant breakthrough in elucidating the role of nuclear remodeling events in reprogramming following SCNT.