Nuclear Reprogramming and Phenotype in Cloned Embryos

描述(申请人提供)：卵母细胞独特地被赋予了将配子基因组转化为胚胎基因组的分子机制。卵母细胞也被赋予了丰富的母体信使核糖核酸(MmRNA)供应，以维持胚胎直到其基因组形成并被激活，这些MmRNAs被招募以精心策划的、特定于阶段的方式进行翻译。体细胞核移植为探索卵质-核相互作用提供了一种创新的工具，可以深入了解启动每个新生命的早期过程，如核重新编程和MmRNA调控。在之前的获奖期间，我们发现克隆胚胎中MmRNA降解的时间模式被打乱，揭示了核在控制MmRNA招募和降解中的新作用。这种缺陷与总蛋白质合成率降低和抑制翻译的EIF4EBP1的过度表达有关。在我们的第一个目标中，我们将表征EIF4EBP1和eIF4E的阶段特异性表达和磷酸化。此外，我们将在克隆和受精胚胎中操纵这些活动，以剖析它们在发育过程中对MmRNA调控的贡献，从而深入了解核是如何控制MmRNA翻译的。我们还发现，在克隆的2-细胞胚胎中，&gt；800基因过度表达。最大类别的过度表达基因编码活跃在供体细胞基因组中的转录因子(TF)及其下游靶基因的整个网络。我们假设，持续表达转录因子的供体细胞库会产生“涟漪效应”，引导许多基因的异常表达，并创造出克隆的许多异常特征。我们还假设供体核中的转录状态预测SCNT后的基因表达。在目标2中，我们将调控两个转录因子(KLF4，CBX4)在克隆胚胎中的表达和活性，并监测其对基因表达的影响，以验证这一假说。我们还将操纵供体细胞和正常胚胎中TF的表达，以确定供体核中的转录状态在克隆胚胎中预期表达的程度。这将为管理核重新编程的参数提供新的见解，并为特定基因网络的实际重新编程成功提供一个定量衡量标准。我们还观察到，在体外可用于将体细胞转化为诱导多能干细胞(iPS细胞)的四个基因中，有两个在克隆中与受精胚胎相比错误表达；Klf4在克隆胚胎中高表达，而Sox2低表达。其中的另一个基因Myc在正常情况下的表达水平很低，可能不足以重新编程。在目标3中，我们将确定用于在体外制造iPS细胞的基因是否也可以用于改善克隆胚胎的表型。目标2和目标3将首次共同解决特定基因重新编程的机制和限制，将我们对“有限重新编程”的理解从一个抽象的概念推进到对受影响的特定分子途径的理解。与克隆胚胎相比，操纵受精胚胎的结果也将为个体发育过程如何控制协调表达的基因网络提供新的见解。

DESCRIPTION (provided by applicant): The oocyte is uniquely endowed with the molecular machinery to convert gamete genomes to an embryonic genome. The oocyte is also endowed with a rich supply of maternal mRNA (MmRNA) to sustain the embryo until its genome is formed and becomes activated, and these MmRNAs are recruited for translation in a carefully orchestrated, stage-specific manner. Somatic cell nuclear transfer provides an innovative tool for exploring ooplasm-nucleus interactions, to gain insight into the early processes that initiate each new life, such as nuclear reprogramming and MmRNA regulation. During the previous award period we discovered that the temporal pattern of MmRNA degradation is disrupted in cloned embryos, revealing a novel role for the nucleus in controlling MmRNA recruitment and degradation. This defect is associated with a reduced total protein synthesis rate and over-expression of EIF4EBP1, which inhibits translation. In our first Aim, we will characterize the stage-specific expression and phosphorylation of EIF4EBP1 and EIF4E. Furthermore, we will manipulate these activities in cloned and fertilized embryos in order to dissect their contributions to MmRNA regulation during development, thereby gaining insight into how the nucleus controls MmRNA translation. We also discovered that >800 genes are over-expressed in cloned 2-cell embryos. The largest category of over-expressed genes encodes transcription factors (TFs) active in the donor cell genome, along with entire networks of their downstream target genes. We hypothesize that persistent expression of the donor cell repertoire of TFs creates a "ripple effect", directing aberrant expression of many genes, and creating the many aberrant characteristics of clones. We also hypothesize that transcription state in the donor nucleus anticipates gene expression after SCNT. In Aim 2, we will manipulate the expression and activities of two TFs (KLF4, CBX4) in cloned embryos and monitor effects on gene expression in order to test this hypothesis. We will also manipulate TF expression in donor cells and in normal embryos to determine the degree to which transcription state in the donor nucleus anticipates expression in the cloned embryo. This will provide new insight into parameters governing nuclear reprogramming, as well as a quantitative measure of actually reprogramming success for specific gene networks. We also observe that two of the four genes that can be used to convert somatic cells to induced pluripotent stem cells (iPS cells) in vitro are mis-expressed in clones compared to fertilized embryos; Klf4 is over-expressed in cloned embryos, while Sox2 is under-expressed. Another of these genes, Myc, is normally expressed at a low level that may be inadequate for reprogramming. In Aim 3 we will determine whether the genes employed to make iPS cells in vitro can also be employed to improve cloned embryo phenotype. Aims 2 and 3 will collectively, for the first time, address mechanisms and limitations of reprogramming of specific genes, advancing our understanding of "limited reprogramming" from an abstract concept to an understanding of specific molecular pathways that are affected. The results of manipulations of fertilized embryos in comparison to cloned embryos will also provide new insight into how networks of coordinately expressed genes are controlled by ontogenetic processes.