A novel epigenetic mechanism in early embryogenesis

早期胚胎发生中的一种新的表观遗传机制

• 批准号: 10799233
• 负责人: zhuo Andrew Xiao
• 金额: $ 19.39万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10799233
• 关键词: Advocate; Binding; Biochemistry; Biological Assay; Cell Culture Techniques; Cells; DNA; DNA Methylation; DNA Modification Methylases; DNA Transposable Elements; Development; Embryo; Embryonic Development; Epigenetic Process; Fertilization in Vitro; Fetus; Gene Expression; Genetic Engineering; Genetic Transcription; Genotype; Investigation; Morula; Mus; Outcome; Proteins; Publishing; Regulatory Pathway; Research; Research Project Grants; Running; Sampling; Time; blastocyst; embryo tissue; embryonic stem cell; experimental study; human disease; instrument; mouse model; novel; ran GTP-Binding Protein

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.

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，如内源性转座因子（TEs）残留的古代 转座子和DNA甲基化。我们使用胚胎干细胞（ESC）培养，遗传- 工程小鼠模型和体外受精（IVF）小鼠模型，以询问 转录和表观遗传调控途径在胚胎干细胞或发育中的小鼠胎儿。 目前的建议主张需要购买一个多功能的实时PCR 机器，以准确地量化基因表达，表观遗传因素和基因型在早期 胚胎和胚外组织（目标1和2），并运行蛋白质热位移测定， 生物化学实验（Aim 3）。 我们近一半的样品是从有限数量的材料中产生的，例如 100-200个来自桑椹胚或胚泡期小鼠胎儿的细胞。我们的目标往往具有挑战性， 例如高度重复的TE。这些独特的研究需求需要一个精确的， 可靠的仪器，其还具有作为正交试验运行Taqman测定的能力。 该方法独立验证基于SYBR green的实时PCR检测的结果。 此外，我们的实验通常需要不同的吞吐量，这需要交换 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孔板中进行常规的基于SYBR绿的测定， 然而，小鼠胚泡中TE表达水平的检查需要Taqman测定， 384-好板。 此外，我们的家长建议的一个主要方向，是研究 SATB 1，一种与DNA结合的关键重塑因子，以及新的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测定的仪器来检查 蛋白质质量和它们与DNA底物的结合，如我们最近发表的研究所示。 本课题旨在研制一种准确、通用、快速的实时PCR仪 满足上述父母提案中多种独特的研究需求。的 该提案的结果将极大地促进我们在表观遗传学方面的研究， 胚胎发生