Identifying Epigenetic, Chromatin, and Transcriptomic landscapes to Improve SCNT Development in an Animal Model

识别表观遗传、染色质和转录组景观以改善动物模型中 SCNT 的发展

基本信息

批准号：
10406300
负责人：
BRADLEY R. CAIRNS
金额：
$ 41.71万
依托单位：
UTAH STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-01 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10406300
关键词：
Agriculture Animal Model Animals Binding Bioinformatics Biopsy Cattle Cell Nucleus Cells Chromatin Clinical Cloning DNA DNA Methylation DNA methylation profiling Data Defect Development Developmental Process Diagnostic Dissection Ectopic Expression Embryo Embryo Cloning Embryo Transfer Enhancers Epigenetic Process Exhibits Failure Female Fibroblasts Gene Expression Generations Genes Genetic Genetic Engineering Genetic Transcription Genome Goals Heart Heterogeneity Human In Vitro Intervention Knowledge Lead Livestock Maps Measures Memory Minority Molecular Nature Nucleic Acid Regulatory Sequences Oocytes Outcome Patients Pre-implantation Embryo Development Process Production RNA Repression Reproduction Research Resistance Specificity Testing Therapeutic Time adverse outcome animal cloning base blastocyst cell type clinical application differential expression egg embryonic stem cell epigenome genome wide methylation genome-wide imprint improved induced pluripotent stem cell innovation insight male novel strategies preimplantation programs promoter prospective screening sex somatic cell nuclear transfer sperm cell success tool transcription factor transcriptomics

项目摘要

PROJECT SUMMARY Somatic cell nuclear transfer (SCNT) allows for the creation of genetically identical animals for agricultural purposes, and is a powerful tool for production of genetically engineered animals for biomedical applications and generation of patient-specific embryonic stem cells (SCNT-ESCs) for therapeutics. However, the low efficiency of full term development limits its wide-spread use and practical utility. The fundamental problem is how the egg incompletely reprograms a somatic donor nucleus. The overall objectives of the current proposal are outlined in the two Specific Aims : 1) Identify epigenetic, chromatin, and transcriptomic landscapes in in vitro fertilized (IVF) embryos and then elucidate how SCNT embryos deviate from this developmental paradigm; and 2) Establish and test new approaches to correct reprogramming defects and improve SCNT development both in preimplantation and postimplantation embryos. We have created high- quality open chromatin maps for carefully-staged IVF and SCNT bovine embryos and leveraged cutting-edge single-cell open chromatin profiling to clarify intra- and inter-embryo heterogeneity in chromatin reprogramming. These maps allow us to interrogate, for the first time, how specific regions of the genome open and close during normal preimplantation development, and how SCNT embryos deviate from this paradigm. Our preliminary data clearly indicate early cleavage staged SCNT embryos do not establish open chromatin properly, and implicate an important contributor to EGA, a transcription factor (TF) DUX, as a major deficiency in SCNT embryos. We hypothesize that failures in chromatin reprogramming during SCNT preimplantation development manifest later in postimplantation development, and that targeted intervention using TF expression or preimplantation screening will improve SCNT developmental success. Aim 1 consists of three subaims: 1.1. Determine sex-specific SCNT open chromatin regions that deviate from IVF embryos, or exhibit sex-specificity; 1.2. Identify somatic memory in SCNT embryos that is resistant to reprogramming using DNA methylation profiling; and 1.3. Identify gene expression differences between SCNT and IVF blastocysts and evaluate if they are biased towards ICM or TE lineages. Based on these data, we will use bioinformatic analyses to identify additional TFs/regulators for further mechanistic dissection. In Aim 2, three subaims will be tested: 2.1. if ectopic enforcement of EGA (using DUX and other TFs) can reactivate the EGA program in cultured fibroblasts; 2.2. If targeted activation of the EGA network in SCNT embryos using TF or TF combinations (from 2.1) would improve reprogramming and facilitate their development and 2.3. If single- cell biopsy and open chromatin profiling at early stages can predict developmental outcomes of both IVF and SCNT embryos. These goals will improve the efficiency of generation of agricultural and biomedical animal models and establish strategies and insights for human clinical applications.

项目概要体细胞核移植（SCNT）可以创造出基因相同的动物农业用途，是生产用于生物医学的基因工程动物的强大工具用于治疗的患者特异性胚胎干细胞（SCNT-ESC）的应用和生成。然而，长期开发效率低限制了其广泛使用和实用。基本的问题是卵子如何不完全地重新编程体细胞供体核。总体目标当前的提案概述为两个具体目标：1) 识别表观遗传、染色质和转录组体外受精 (IVF) 胚胎的景观，然后阐明 SCNT 胚胎如何偏离此景观发展范式； 2) 建立并测试纠正重编程缺陷的新方法改善植入前和植入后胚胎的 SCNT 发育。我们创造了高用于精心分阶段的 IVF 和 SCNT 牛胚胎的高质量开放染色质图谱，并利用尖端技术单细胞开放染色质分析，以阐明染色质的胚胎内和胚胎间异质性重新编程。这些地图让我们第一次询问特定区域如何正常植入前发育过程中基因组的打开和关闭，以及 SCNT 胚胎如何偏离从这个范式。我们的初步数据清楚地表明早期卵裂阶段的 SCNT 胚胎不正确建立开放染色质，并暗示 EGA 的重要贡献者，一种转录因子 (TF) DUX，是 SCNT 胚胎的一个主要缺陷。我们假设染色质重编程失败在 SCNT 植入前发育期间，随后在植入后发育中显现出来，并且目标是使用 TF 表达或植入前筛查进行干预将提高 SCNT 的发育成功率。目的 1 由三个分目标组成： 1.1.确定偏离 IVF 的性别特异性 SCNT 开放染色质区域胚胎，或表现出性别特异性； 1.2.鉴定 SCNT 胚胎中具有抗性的体细胞记忆使用 DNA 甲基化分析进行重编程；和1.3。识别 SCNT 之间的基因表达差异和 IVF 囊胚，并评估它们是否偏向 ICM 或 TE 谱系。根据这些数据，我们将使用生物信息学分析来识别额外的转录因子/调节因子，以进行进一步的机制剖析。在目标 2 中，将测试三个子目标： 2.1.如果 EGA 的异位执行（使用 DUX 和其他 TF）可以重新激活培养成纤维细胞的 EGA 程序； 2.2.如果使用 TF 有针对性地激活 SCNT 胚胎中的 EGA 网络或 TF 组合（来自 2.1）将改善重编程并促进其发展，2.3。如果单- 早期细胞活检和开放染色质分析可以预测 IVF 和 IVF 的发育结果 SCNT 胚胎。这些目标将提高农业和生物医学动物的生产效率模型并建立人类临床应用的策略和见解。