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 not to prematurely activate during fusion, despite the presence of calcium in the fusion medium [23, 24]. It is likely that there are differences between species with the ease at which recipient cytoplasts activate upon fusion. Other factors such as the age of recipient cytoplasts, source of oocytes (in vivo-matured or in vitro-matured), and fusion parameters utilized are also likely to influence the activation status of fused cybrids.暴露在未激活的卵母细胞的细胞质中被认为是促进供体核的重塑和重新编程，[12，22]。
未激活细胞质中含有活跃的成熟促进因子（MPF），它能诱导核膜破裂（NEBD）和移植核的染色体提早浓缩。
当不考虑供体细胞中细胞周期的存在时，牛卵丘细胞与去核的卵子融合期间，，谷等人[9]，最近观察到核膜破裂和染色体提早浓缩。
在目前的研究中，为避免在大多数受体细胞质中发生并发激活，我们证明了在缺钙条件下培养对于融合供体细胞的重要性。
当69%的胞质杂合体融合在含钙的介质中，并不受化学激活的刺激，它们会在培养两天之后分裂，与之相比的是只有10%的类似物融合在缺钙条件下。
以上这些在第一个实验中是很明显的。
尽管在融合介质中有钙的存在，我们发现对照组的结果是，牛体中融合期间重建的胞质杂合体不会过早地活化[23，24]。
有可能在物种间通过融合使受体细胞质的活化有差异。
例如受体细胞质的年龄，卵母细胞的来源（体内成熟或体外成熟），以及融合参数的使用这些因素，都有可能影响到融合杂合体的激活状态。
Cloned offspring from somatic cells have been produced previously as a result of reconstructing embryos either by activating simultaneously during introduction of donor nuclei into recipient cytoplasts [1] or sometime thereafter [3, 11]. A direct comparison between the developmental competence of bovine nuclear transfer embryos by Wells and coworkers [11] found that twice as many (39.8%) embryos reconstructed by fusing cumulus cells into recipient cytoplasts before activation using ionomycin/6-DMAP developed to blastocyst stage in vitro compared with those reconstructed using simultaneous fusion/activation (18.6%). This may be related to higher expression of interferon tau in cloned embryos produced by simultaneous fusion/activation [25], which has been correlated with poor embryo quality in cattle [26]. Here in the pig, we demonstrate, retrospectively, a greatly improved rate of development to blastocyst stage (23%) by activating cybrids 3 h after fusion compared with previous work in our laboratory in which simultaneous fusion/activation was employed (3%; [15]). However, differences between the past study [15] and the current study, including oocyte quality, donor cell line used, activation stimulus (electrical pulse vs. chemical), and donor cell cycle synchronization treatment (serum starvation vs. culture to confluency) were also likely to affect cloning efficiency. Therefore, a direct comparison between both procedures will need to be made to validate whether cloning efficiency in pigs is increased by delaying activation after donor cell fusion.因为通过在向受体细胞质引入供体核是同时激活或者之后的方法来重建胚胎，所以来自体细胞的克隆后代之前已经产生[3，11]。