胚胎翻译 DISCUSSION.docVIP

Discussion 讨论 In the current study, we report the birth of live cloned piglets from cultures of fetal fibroblast cells. These cells were harvested from Day 25 fetuses before being frozen in liquid nitrogen for 2 yr. They were then passaged up to 12 times (approximately 40 population doublings) before nuclear transfer. Taken together, this result illustrates the utility of cloning for storing and multiplying genotypes and for transgenesis by enabling enough population doublings for selection of gene-targeted cells before nuclear transfer. We believe our success was due to the development of a porcine nuclear transfer system in which donor nuclei were exposed to unactivated cytoplasm for approximately 3 h before activation by chemical means. 在目前的研究中，我们报告了通过胚胎成纤维细胞培养出生的克隆活仔猪。这些收集的细胞来自在液氮中冷冻了两年的Day 25 胎儿。然后，在核移植前他们传代12次（约40倍群体倍增）。两者合计，为储存和繁殖基因型的克隆，并且在核移植前选择基因靶标细胞来确保足够的群体倍增和进行转基因组的克隆，结果表明这两者都是可利用的。我们相信，我们的成功是由于猪核移植系统的发展，它是用化学方法激活之前移植核供体暴露在未活化的细胞质中大约三个小时。 Exposure to unactivated oocyte cytoplasm is believed to facilitate remodeling and reprogramming of donor nuclei, [12, 22]. Unactivated cytoplasm contains high levels of active maturation promoting factor (MPF), which induces nuclear membrane breakdown (NEBD) and premature chromatin condensation (PCC) of transferred nuclei. Tani et al. [9] recently observed NEBD and PCC when bovine cumulus cells were fused with enucleated ova, regardless of the phase of the cell cycle in which donor cells existed. In the current study, we demonstrate the importance of fusing donor cells under calcium-free conditions to avoid concurrent activation in the majority of recipient cytoplasts. This was evident in the first experiment when 69% of cybrids that were fused in calcium-containing medium and not receiving chemical activation stimulus cleaved after 2 days of culture compared with only 10% of their counterparts fused under calcium-free conditions. Our finding contrasts with results in cattle in which reconstructed cybrids were found n