Altered Nuclear Transfer

描述(申请人提供)：这个项目的目的是产生关于卵母细胞表观遗传因素的重要新见解，这些因素支配着体细胞核移植(SCNT)后的重编程，以及哺乳动物胚胎中前两个谱系--内细胞团(ICM)和滋养外胚层(TE)的形成和维持。我们的主要假设是，对卵母细胞中关键转录因子表达水平的实验调节将防止TE的形成，同时促进ICM谱系的产生。这些改变应该阻止胚胎的形成，同时促进和加强供体核重新编程，这是形成单一谱系所需的，从这个谱系可以建立多能干细胞系。我们提出了以下具体目标：目的1.建立一种改进的ESC衍生法建立蚂蚁模型。首先，我们将研究siRNA介导的小鼠卵母细胞中CDX2、Tead4和E-钙粘附素基因敲除对SCNT后体细胞重编程的影响。接下来，我们将研究TE失活以及关键多能因子-Oct4、Sox2和Nanog-上调对ESCs重新编程和衍生的累积影响。目的2.明确猕猴卵母细胞中对TE发育至关重要的因素。在实验1中，我们将检测TEAD4在猴子卵母细胞和植入前胚胎中的存在和表达谱。接下来，我们将通过吗啡辅助敲除来研究TEAD4在猴子卵母细胞和胚胎中的作用。最后，受精后的TEAD4缺陷卵母细胞将用于胚胎干细胞分离。目的：分析关键的母体TE因子在猴SCNT后表遗传学重编程中的作用。为此，我们将应用最有希望的方法来评估ANT在猴子模型中的有效性，方法是首先创建CDX2和TEAD4耗尽的卵母细胞，然后进行SCNT，然后从得到的多能细胞中获得ESCs。

DESCRIPTION (provided by applicant): The aim of this project is to generate important new insights concerning epigenetic factors in oocytes governing reprogramming following somatic cell nuclear transfer (SCNT) and formation and maintenance of the first two lineages in mammalian embryos, the inner cell mass (ICM) and trophectoderm (TE). Our main hypothesis is that experimental modulation of expression levels for the key transcription factors in oocytes will prevent formation of the TE while enhancing the production of the ICM lineage. These alterations should preclude the formation of an embryo while promoting and enhancing donor nucleus reprogramming required for the formation of a single lineage from which pluripotent stem cell lines can be established. We propose the following specific aims: Aim 1. Establish a mouse model of ANT with improved ESC derivation. Initially, we will investigate the effect of siRNA-mediated knockdown of Cdx2, Tead4, and E-Cadherin in mouse oocytes on reprogramming of somatic cells after SCNT. Next, we will examine a cumulative impact of the TE inactivation along with upregulation of critical pluripotent factors - Oct4, Sox2, and Nanog - on reprogramming and derivation of ESCs. Aim 2. Define factors in monkey oocytes that are crucial for TE development. In Experiment 1, we will examine the presence and expression profiles of TEAD4 in monkey oocytes and preimplantation embryos. Next, we will study the role of TEAD4 in monkey oocytes and embryos by morpholino-assisted knockdown. Lastly, fertilized TEAD4-deficient oocytes will be used for ESC isolation. Aim 3: Analyze the roles of key maternal TE factors in epigenetic reprogramming after monkey SCNT. In this aim, we will apply most promising approaches to assess the validity of ANT in the monkey model by first creating CDX2 and TEAD4 depleted oocytes and then conducting SCNT and subsequently deriving ESCs from the resultant pluripotent cells.