A novel epigenetic mechanism in early embryogenesis

我们的父母建议旨在研究哺乳动物早期的关键机制 胚胎发生，如古代残留的内源性转座元件(TES) 转座子和DNA甲基化。我们使用胚胎干细胞(ESC)培养，遗传- 基因工程小鼠模型和体外受精(IVF)小鼠模型 胚胎干细胞或发育中的小鼠胚胎的转录和表观遗传调控途径。 目前的提案主张需要购买多功能的实时聚合酶链式反应 机器在早期准确地量化基因表达、表观遗传因素和基因型 胚胎和胚外组织(目标1和2)和Run蛋白热位移分析 生化实验(Aim3)。 我们近一半的样品是由有限的材料产生的，例如 100-200个细胞来自桑拿期或囊胚期的小鼠胚胎。我们的目标往往是具有挑战性的， 例如高度重复性的TES。这些独特的研究需求要求精确和 可靠的仪器，也有能力运行塔克曼分析作为一个正交表 一种独立验证SYBR Green实时荧光聚合酶链式反应检测结果的方法。 此外，我们的实验经常需要不同的吞吐量，需要交换 在96孔板和384孔板之间。例如，基因表达的常规检查 胚胎干细胞的水平需要在96孔板上进行基于SYBR-Green的常规检测， 而检测小鼠胚泡中TE的表达水平需要Taqman分析 384井板。 此外，我们的父母建议的一个主要方向是调查 与DNA结合的关键重塑因子SATB1和新的DNA甲基转移酶 (目标3)。因此，我们需要一种可以运行蛋白质热位移分析的仪器来进行检查 蛋白质质量及其与DNA底物的结合，如我们最近发表的研究所示。 本方案的目的是获得一台准确、通用、快速的实时荧光PCR机 这满足了上面概述的父母提案中的多种独特研究需求。这个 这项建议的结果将极大地促进我们在表观遗传学和 胚胎发生。

Our parental proposal aims to investigate critical mechanisms in mammalian early embryogenesis, such as endogenous transposable elements (TEs) remnants of ancient transposons and DNA methylation. We use embryonic stem cell (ESC) culture, genetic- engineered mouse model and in vitro fertilization (IVF) mouse models to interrogate the transcription and epigenetic regulatory pathways in ESC or developing mouse fetus. The current proposal advocates the needs for purchasing a versatile real-time PCR machine to accurately quantify the gene expression, epigenetic factors and genotype in early embryonic and extraembryonic tissues (Aim 1 and 2) and run protein thermal shift assays for biochemistry experiments (Aim3). Nearly half of our samples are generated from limited amounts of materials, such as 100-200 cells from morula or blastocyst stage mouse fetus. Our targets are often challenging, such as TEs that are highly repetitive. These unique research needs demand a precise and reliable instrument which also has the capability to run the Taqman assays as an orthogonal approach to independently validate the results from SYBR green-based real-time PCR assays. Furthermore, our experiments often require different throughputs, which need to interchange between a 96-well and 384-well plates. For example, a routine examination of gene expression level in embryonic stem cells needs a routine SYBR-green based assay in 96-well plates, whereas an examination of TE expression levels in mouse blastocyst needs Taqman assays in 384-well plates. Moreover, a major direction of our parental proposal is to investigate the function of SATB1, a critical remodeling factors binding to DNA, as well as novel DNA methyltransferases (Aim3). Thus, we need to an instrument that can run protein thermal shift assays to check protein quality and their binding to DNA substrates as shown in our recently published studies. This proposal aims to acquire an accurate, versatile and fast Real-Time PCR machine that meets multiple unique research needs in the parental proposal outlined above. The outcome of the proposal will greatly facilitate our investigation in epigenetics and embryogenesis.