胚胎翻译 DISCUSSION

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]。

通过威尔和他的同事创立的牛核移植胚胎感受态的发展，他们发现与用同步融合法的重建相比（18.6％），在融合前用伊屋诺霉素活化在体外发展为囊胚期，通过融合卵丘细胞进入受体细胞质而进行的胚胎重建，后者是前者的两倍(39.8%)。

这可能与用同步融合产生的胚胎中干扰素的较高表达有关，这与牛胚胎质量差有关[26]。

在猪体内我们已经证明，与我们之前实验室中运用的同步融合法比，通过融合后刺激杂合体3个小时，囊胚期发生率有更大的提高[15]。

然而，在过去与现在的研究中，包括卵母质量，供体细胞系的使用，活化刺激和供体细胞周期同步的方法，这些不同很有可能会影响到克隆效率。

为此，两种方法的直接对比需要验证通过看用延迟激活供体细胞融合后猪体内克隆效率是否增加。

Introduction of donor nuclei into recipient cytoplasts is arguably the most difficult step in cloning by nuclear transfer. Efficient reconstruction is particularly vital in pig cloning because of the necessity to transfer large numbers of cloned embryos into each recipient to ensure the presence of a critical number of fetuses for pregnancy maintenance. Therefore, we increased numbers of cloned embryos available for
transfer by exposing couplets to a second round of fusion if they remained unfused after the initial electrical pulse application. We justified recycling unfused couplets because we observed that embryos reconstructed after a second pulse were found to develop to blastocyst in vitro at a similar rate to their counterparts successfully fused after the first pulse (data not shown). However, it is unknown whether application of one or two pulses affected subsequent in vivo development of cloned embryos.
通过核移植，供体核向受体细胞质中的引入是克隆中最难的一步。

维持妊娠确保一定数量胎儿的存在，因为转移大量克隆胚胎进入受体很有必要，所以猪克隆中有效的重建很重要。

为此，我们应增加大量可用的克隆胚胎，如果细胞在第一次电刺激中未融合，可以通过把他们暴露在二次融合中解决。

我们循环使用未融合的偶联体，因为我们发现二次刺激的胚胎重建体外发展成囊胚体的速度与一次成功的一样。

然而，一次或两次的刺激是否会影响体内克隆胚胎的发展现在还不知道。

Previous pig cloning studies resulting in full-term development primarily differ in the reconstruction procedure employed (microinjection [5], electrofusion [7], or serial nuclear transfer [6]) as well as the cell type (cumulus [6], fetal fibroblast [5, 7], or genital ridge [18]) and recipient cytoplast (in vitro-matured [7] or in vivo-matured [5]) used. Themes common to these studies and the present report include the transfer of cloned embryos into uteri of recipients after 3 days of culture as well as the use of donor cells cultured to confluency, which is likely to synchronize cells in G1 [19] and may avoid DNA fragmentation as seen in serum-starved cells [27]. Our study is most similar to that by Onishi and coworkers [5] because fetal fibroblast cells were introduced into in vivo-flushed oocytes without concurrent activation, then activated a few hours thereafter. A differing aspect is that Onishi et al. [5] microinjected donor nuclei, whereas we introduced the entire donor cell contents by electrofusion, although the procedure used to introduce nuclei may not affect development because Ogura et al. [28] found similar in vitro and in vivo rates of development in cloned mice regardless of whether microinjection or electrofusion was used. In the study by Betthauser et al. [7], donor nuclei were fused into enucleated in vitro-matured sow oocytes, then, like in the present study, cybrids were chemically activated by ionomycin/6-DMAP. However, based on the data presented in the current study, it is possible that the majority of embryos produced by Betthauser et al. [7] may have prematurely activated because of the presence of calcium in the fusion medium. Unlike in the current study, the activation status of a cohort of cybrids not exposed to chemical activation was not determined. As with in vivo oocytes, we have recently found that when enucleated in vitro-matured sow oocytes are used, fusion in calcium-free medium is essential to avoid concurrent activation (unpublished result). 造成长远发展的前猪克隆研究主要在重建过程中不同（显微注射[5]，电熔[7]，或串行核转移[6]），以及细胞类型（积云[6]，胎儿成纤维细胞[5,7]，或生殖嵴[18]）和收件人胞质（在体外成熟[7]或在体内成熟[5]）使用。

这些研究和本报告的共同主题，包括到受助人子宫的克隆胚胎转移后3天的文化，以及利用体细胞培养的汇合，这可能是同步细胞在G1 [19]，可避免在血清饥饿的细胞的DNA片段[27]。

我们的研究是最相似的（音译）和他的同事[5]
引入，因为胎儿成纤维细胞在体内刷新的卵母细胞无并发激活，然后激活后几个小时。

不同的方面是，大西等。

[5]显微注射供体核，而我们介绍了由电熔整个供体细胞的内容，虽然用于引进原子核的过程可能不会影响发展的，因为小仓等。

[28]发现在体外和体内的发展率，在克隆小鼠，无论是否使用显微注射或电熔类似。

Betthauser等人的研究。

[7]，供体核到去核体外成熟的母猪卵母细胞融合，然后，像在本研究中，杂合体是化学伊屋诺霉素激活。

然而，根据目前的研究中提出的数据，它可能是大多数Betthauser等生产的胚胎。

[7]可能有过早，因为在融合液中的钙激活。

在目前的研究不同，不暴露在化学活化杂合体群激活状态不能确定。

在体内卵母细胞，我们最近发现，在体外成熟的母猪卵母细胞去核时，用于的，在无钙媒介融合是必不可少的，以避免并发激活（未发表结果）。

In the current study, development of cloned embryos in vitro was high (23%) compared with the study by Betthauser et al. [7] (7%), but it was not reported by Onishi et al. [5] or Polejaeva et al. [6]. We also found the rate of pregnancy initiation after transfer of cloned embryos into recipients was high (50%; 5 initiations out of 10 recipients) compared with other pig cloning studies, including Betthauser et al. [7] (23%; 15 out of 66) and Polejaeva et al. [6] (29%; 2 out of 7), but cannot be compared with Onishi et al. [5], who did not confirm pregnancy status by ultrasound. Because enucleation was unconfirmed by epifluorescence (‘‘blind enucleation’’) in our study, it is likely that some of the embryos produced were the result of parthenogenetic development. However, because in previous experiments we found the average efficiency of blind enucleation was high (88%), parthenogenetic development would have had only a minimal effect on in vitro development and pregnancy outcomes in the current report.
在目前的研究，克隆胚胎在体外（23％）相比Betthauser等人（7％）的研究发展高[7]，但它不是由Onishi或Polejaeva等报道的[6]。

我们还发现，克隆胚胎转移到受助后开始怀孕率较高（50％;5发起了10个收件人）相比，与其他猪的克隆研究，包括Betthauser等。

[7]（占23％;1566）和Polejaeva等（29％;27），但不能与大西等人相比。

通过超声波谁没有确认怀孕状况。

由于眼球摘除在我们的研究（“盲眼球摘除”）的未经证实的，它是可能的，所产生的胚胎一些孤雌发育的结果。

然而，因为在以前的实验中，我们发现盲目眼球摘除的平均效率高（88％），孤雌发育会曾在体外发育和妊娠结果，在当前的报告只影响甚微。

In this study, we produced cloned piglets from cultures of fetal fibroblast cells that had previously been stored in liquid nitrogen for 2 yr. The improvement in pig cloning efficiency here, in comparison to our previous study [15], is likely to be the result of exposing nuclei to unactivated cytoplasm for a few hours before chemical activation. To achieve this, we demonstrate the necessity to fuse donor cells with recipient cytoplasts in calcium-free medium.
Using the cloning technique described here, our efforts are now focused on producing a(1, 3)-galactosyltransferase gene knock-out pigs for the possible future use in human transplantation.
在这个研究中，我们用已在液氮中储存了两年的胚胎成纤维细胞培养从而产生了克隆小猪。

比起我们之前的研究，这次克隆小猪效率的提高很有可能是因为在化学活化之前将细胞核暴
露在细胞质中几个小时。

完成这个实验，我们证明了用受体细胞质和供体细胞在缺钙介质中的融合是很重要的。

运用以上所介绍的克隆技术，我们现在致力于研究半乳糖转移酶基因敲除的猪，它在未来人体移植中有可能有用。