Functional Annotation of the Preimplantation Transcriptome

描述(申请人提供)：植入前转录组的功能注释。这项建议的主要目标是阐明在哺乳动物植入前发育过程中表达的大量基因的需求。在RNAi介导的每个基因功能丧失后，我们将评估形态发育、谱系指定、基因组印迹的表观遗传调控和胚胎干细胞衍生潜力。这项工作将扩展我们已经进行的试点RNAi筛查(~500个基因)，以便为整个转录组的努力建立条件。当前存在足够的数据集，使得 我们不需要确定哪些基因是自己表达的--但可以利用其他小组的工作，直接向我们的目标--植入前转录组的功能注释--发起。我们的系统利用了受精后最初几天发生的动态基因组重新编程。我们的方法使用小鼠胚胎作为活体模型系统，并监测早期发育事件和基因组印记。我们的方法为识别新的表观遗传调控机制以及发育过程中的功能需求提供了一种稳健而有效的方法，我们完全准备开展拟议的工作。除了我们的数据将提供的基本科学知识之外，我们的建议还与理解证据高度相关，这些证据表明，即使在植入前短期接触体外培养也可能产生可怕的表观遗传后果。也许更相关的是通过使用人工生殖技术在单个哺乳动物的胚胎和胎盘中产生表观遗传缺陷的许多例子。了解植入前调控表观遗传事件的基本分子机制是至关重要的，如果我们要改进目前的实践，以及设计针对许多人类疾病状态的治疗方法，这些疾病存在表观基因表达的错误调节。

DESCRIPTION (provided by applicant): Functional Annotation of the Preimplantation Transcriptome. The primary goal of this proposal is to elucidate the requirement of a large set of genes expressed during mammalian preimplantation development. After RNAi mediated loss of each gene function, we will assess morphological development, lineage specification, epigenetic regulation of genome imprinting and embryonic stem cell derivation potential. This work will extend a pilot RNAi screen that we have performed (~500 genes) in order to establish conditions for this transcriptome-wide effort. Currently there exist sufficient data sets such that we will not need to determine which genes are expressed ourselves - but can use the work of other groups in order to launch directly towards our goal of functional annotation of the preimplantation transcriptome. Our system capitalizes on the dynamic genome reprogramming that occurs during the first few days post-fertilization. Our approach uses mouse embryos as an in vivo model system and monitors early developmental events and genomic imprinting. Our approach provides a robust and efficient method towards identification of novel epigenetic regulatory mechanisms as well as functional requirements during development and we are uniquely poised to carry out the proposed work. In addition to the bolus of basic scientific knowledge our data will provide, our proposal is highly relevant for understanding the evidence that even short-term exposure to in vitro culture during preimplantation can have dire epigenetic consequences. Perhaps even more relevant are numerous examples of epigenetic defects in embryos and placentas of individual mammals generated through the use of artificial reproductive technologies. Understanding the basic molecular machinery that regulates epigenetic events during preimplantation is of critical importance if we are to improve current practice as well as to design therapeutic approaches for the many human disease states with epigenetic mis-regulation of gene expression.