Mechanism for the reprogramming of gene expression in the oocytes and preimplantation embryos

本研究项目包括以下3个步骤。1细胞期合子基因激活（ZGA）开始时表达的基因图谱。通过转录因子和表观遗传修饰分析生长中的卵母细胞和早期着床前胚胎的基因表达调控。rnai功能分析这些研究的结果描述如下1。用BrUTP标记分离新生mrna，用抗bru抗体免疫沉淀，然后用微阵列分析。在1细胞阶段鉴定出50多个基因为ZGA基因。对这些基因调控区域的分析表明，其中很大一部分具有Pax和Ets家族转录因子结合的共识序列2。1500个转录因子基因的基因芯片分析表明，Ets家族转录因子的表达模式在1细胞和2细胞阶段变化最大，受精后表达量显著增加。免疫细胞化学对组蛋白修饰的分析表明，在卵母细胞的生长过程中，所有的修饰都增加了，并且在种植前发育过程中，许多修饰以不同的模式发生变化。将生长中的卵母细胞微注射RNAi，使其在体外发育为完全成熟的卵母细胞，进行减数分裂成熟和着床前发育。我们证实这个系统可以很好地敲除一个特定的基因，并分析其对卵母细胞和胚胎发育的作用。

This research project consists of following 3 steps.1. Profiling of the genes which are expressed at the onset of zygotic gene activation (ZGA) at 1-cell stage.2. Analysis for the regulation of gene expression in the growing oocytes and early preimplantation embryos by examining transcription factors and epigenetic modifications.3. Functional analysis by RNAiThe results of these studies are described below1. The nascent mRNAs were isolated by being labeled with BrUTP and immunoprecipitated with anti-BrU antibody, and then analyzed by microarray. More than 50 genes were identified as ZGA genes at 1-cell stage. The analysis of the regulatory regions of these genes revealed that a large part of them have the consensus sequences to which Pax and Ets family transcription factors bind.2. Analysis by a microarray of 1500 transcription factor genes showed that the expression patterns most changed between 1-cell and 2-cell stage and that the expression of Ets family transcription factors greatly increased after fertilization. Analysis for histone modifications by immunocytochemistny revealed that all modifications examined increased during growth of oocytes and that many modifications changed in various patterns during pre-plantation development.3. The growing oocytes were microinjected with RNAi and allowed to growth into full-grown oocytes, meiotic maturation and preimplantation development in vitro. We conformed that this system works well to knock-down a particular gene and analyze its function for the development of oocytes and preimplantaion embryos.