Wakayama Sayaka, Cibelli Jose B, Wakayama Teruhiko
Advanced Cell Technology, Worcester, Massachusetts, USA.
Cloning Stem Cells. 2003;5(3):181-9. doi: 10.1089/153623003769645848.
Cloning methods are now well described and becoming routine. Yet the frequency at which live cloned offspring are produced (as a percentage of starting one-cell embryos) remains below 5% irrespective of nucleus donor species or cell type. In considering the cause(s) of this universally low efficiency, features common to all cloning protocols are strong candidates. One such shared feature is enucleation; the donor nucleus is inserted into an enucleated cytoplast (ooplast). However, it is not known whether a nucleus-free metaphase II oocyte is developmentally impaired other than by virtue of lacking chromosomes, or if in nuclear transfer protocols, enucleation removes factors necessary to reprogram the incoming nucleus. We have here investigated the role of enucleation in nuclear transfer. Three hours after the injection of cumulus cell nuclei into non-enucleated oocytes, 65% contained two distinct metaphase spindles, with the remainder exhibiting a single spindle in which oocyte-derived and nucleus donor chromosomes were mixed. However, staining only one hour after donor nucleus insertion revealed that most had two discrete spindles. In the absence of staining, the donor nucleus spindle was not visible. This provided a straightforward way to identify and select the oocyte-derived metaphase chromosomes 1 h after donor nucleus microinjection, and 34-41% cloned embryo developed to the morulla-blastocyst stage following Sr(2+)-induced activation. Of these, two (1% of starting one-cell embryos) developed to term, an efficiency which is comparable to that obtained for controls (6 clone; 1-2%) in which enucleation preceded nuclear transfer. In conclusion, the timing of the removal of oocyte chromosomes before or after injection of somatic nucleus had no effect on cloned embryo development. These findings argue that neither oocyte chromosome depletion per se, nor the potential removal of "reprogramming" factors during enucleation explain the low efficiency of nuclear transfer cloning.
克隆方法现已得到充分描述并日趋常规化。然而，无论核供体物种或细胞类型如何，活克隆后代的产生频率（以起始单细胞胚胎的百分比计）仍低于5%。在考虑造成这种普遍低效的原因时，所有克隆方案共有的特征是强有力的候选因素。其中一个共同特征是去核；将供体细胞核插入去核的细胞质体（卵质体）中。然而，尚不清楚无核的中期II卵母细胞除了缺乏染色体外是否在发育上受损，或者在核移植方案中，去核是否去除了重编程导入细胞核所需的因子。我们在此研究了去核在核移植中的作用。将卵丘细胞核注入未去核的卵母细胞3小时后，65%含有两个不同的中期纺锤体，其余的则呈现单个纺锤体，其中卵母细胞来源的染色体和细胞核供体染色体混合在一起。然而，在供体细胞核插入仅1小时后染色显示，大多数有两个离散的纺锤体。在没有染色的情况下，供体细胞核纺锤体不可见。这提供了一种直接的方法，在供体细胞核显微注射1小时后识别和选择卵母细胞来源的中期染色体，并且34 - 41%的克隆胚胎在Sr(2+)诱导激活后发育到桑葚胚 - 囊胚阶段。其中，有两个（占起始单细胞胚胎的1%）发育至足月，这一效率与在核移植前进行去核的对照组（6个克隆；1 - 2%）相当。总之，在注入体细胞核之前或之后去除卵母细胞染色体的时间对克隆胚胎发育没有影响。这些发现表明，卵母细胞染色体的缺失本身以及去核过程中“重编程”因子的潜在去除都不能解释核移植克隆的低效性。
