Nuclear Reprogramming and Phenotype in Cloned Embryos

描述(申请人提供)：体细胞核移植克隆为胚胎学的基础研究、干细胞疗法的发展和重要的农业应用提供了令人兴奋的新可能性。克隆仍然非常低效，最近的研究表明，克隆胚胎的核重新编程可能是有缺陷的。然而，没有对重编程进行详细的研究，关于早期胚胎中不完全重编程的表型效应的信息也很少。我们发现，早期克隆的小鼠胚胎在培养介质偏好和DNA甲基转移酶表达方面与正常受精胚胎有很大的不同，相对于对照胚胎表现出更高的葡萄糖摄取，并且缺乏转录后基因调控。其中一些差异甚至在第一次卵裂分裂之前就可以看到，这表明供体核对克隆胚胎的特性有直接影响。我们假设，移植后体细胞基因组的持续表达，结合存在于卵母细胞中的母体mRNAs的翻译，在克隆胚胎中产生介于体细胞和胚胎之间的基因表达谱。这种基因表达模式的改变可能导致克隆胚胎在基本生理和代谢参数方面与正常胚胎明显不同。这可能会导致体内pH调节、渗透调节、动态平衡和ATP产生等基本功能的缺陷。我们还假设，由于这种异常的代谢或生理状态，典型的小鼠胚胎培养环境严重不适合克隆胚胎，因此克隆胚胎存在于一种不利于有效核重新编程的健康状态，导致整体成功的低效率。事实上，我们发现克隆胚胎表现出了惊人的不同培养要求。最后，我们假设不同的体细胞类型可能由于初始供体细胞状态的不同而与克隆过程的兼容性不同。为了检验这些假设，我们将追求三个相辅相成的基本目标。首先，我们将确定被转移的体细胞核在多大程度上继续表达其预先编程的基因库。其次，我们将确定这些核在多大程度上可以指导胚胎特异的基因表达模式，包括适当的转录后招募材料mRNA。第三，我们将确定克隆胚胎中细胞功能的特定代谢和生理方面发生了多大程度的变化。这些目标的实现将为正常胚胎的基本调控和动态平衡机制提供新的信息，也将为进一步研究重新编程的分子基础和提高克隆成功率奠定基础。

DESCRIPTION (provided by applicant): Cloning by somatic cell nuclear transfer offers exciting new possibilities for basic research in embryology, development of stem cell therapies, and important agricultural applications. Cloning remains highly inefficient, and recent studies indicate that nuclear reprogramming may be defective in cloned embryos. However, no detailed study of reprogramming has been undertaken, and little information has been obtained about the phenotypic effects of incomplete reprogramming in the early embryo. We have found that early cloned mouse embryos differ substantially from normal fertilized embryos with respect to culture medium preference and DNA methyltransferase expression, exhibit enhanced glucose uptake relative to control embryos, and are deficient in post-transcriptional gene regulation. Some of these differences can be seen even before the first cleavage division, indicating an immediate effect of the donor nucleus on cloned embryo properties. We hypothesize that continued expression of the somatic cell genome after transfer, combined with the translation of maternal mRNAs present in the oocyte, generates a gene expression repertoire in the cloned embryo intermediate between that of a somatic cell and that of an embryo. This altered gene expression pattern may cause cloned embryos to differ markedly from normal embryos with regard to basic physiological and metabolic parameters. This may lead to defects in such basic functions as internal pH regulation, osmoregulation, homeostasis, and ATP production. We also hypothesize that as a result of this aberrant metabolic or physiological state, typical mouse embryo culture environments are grossly sub-optimal for the cloned embryo, and as a result the cloned embryo exists in a state of poor health that is not conducive to efficient nuclear reprogramming, leading to the low efficiency of overall success. Indeed, we find strikingly different culture requirements manifested by cloned embryos. Last, we hypothesize that different somatic cell types may differ in compatibility with the cloning process due to differences in initial donor cell state. To test these hypotheses, we will pursue three complementary and essential Aims. First, we will determine to what degree the transferred somatic cell nucleus continues to express its pre-programmed repertoire of genes. Second, we will determine to what degree these nuclei can direct embryo-specific gene expression patterns, including appropriate posttranscriptional recruitment of matemal mRNA. Third, we will determine to what degree specific metabolic and physiological aspects of cell function are altered in cloned embryos. Fulfillment of these Aims will provide novel information about basic regulatory and homeostatic mechanisms of normal embryos, and will also a basis for further studies examining the molecular basis of reprogramming, and for improving cloning success.