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：
10187618
负责人：
BRADLEY R. CAIRNS
金额：
$ 45.9万
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/10187618
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.

项目摘要体细胞核转移（SCNT）允许创建遗传相同的动物农业目的，是生产生物医学的基因工程动物的强大工具用于治疗剂的患者特异性胚胎干细胞（SCNT-ESC）的应用和生成。然而，全学期开发的低效率限制了其广泛的使用和实用性。基本问题是鸡蛋如何不完全重新编程体细胞供体核。总体目标当前的建议在两个具体目的中概述：1）识别表观遗传，染色质和转录组。体外受精（IVF）胚胎中的景观发展范式； 2）建立和测试新方法以纠正重编程缺陷和改善植入前和植入后胚胎中的SCNT发育。我们创建了高 - 优质的开放染色质图，用于精心阶段的IVF和SCNT牛胚胎以及杠杆尖端单细胞开放染色质分析，以阐明染色质中的内部和胚胎间异质性重新编程。这些地图使我们能够首次询问基因组在正常植入前开发期间开放和关闭，以及SCNT胚胎如何偏差从这个范式。我们的初步数据清楚地表明早期裂解上演SCNT胚胎没有正确建立开放的染色质，并暗示了EGA的重要贡献，转录因子（TF） DUX，是SCNT胚胎的主要缺陷。我们假设染色质重编程中的失败在SCNT植入前的发展期间，在植入后发展后期表现出来，而这是针对性的使用TF表达或植入前筛查的干预将改善SCNT的发展成功。目的 1由三个子组成：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。识别具有抗性的SCNT胚胎中的体细胞记忆使用DNA甲基化分析重编程；和1.3。识别SCNT之间的基因表达差异和IVF胚泡并评估它们是否偏向ICM或TE谱系。基于这些数据，我们将使用生物信息学分析来识别其他TFS/调节剂以进行进一步的机械解剖。在AIM 2中，将测试三个Subiaim：2.1。如果EGA的异位执行（使用DUX和其他TF）可以重新激活 EGA培养的成纤维细胞计划； 2.2。如果使用TF在SCNT胚胎中的EGA网络有针对性激活或TF组合（从2.1起）将改善重编程并促进其开发和2.3。如果单一细胞活检和开放染色质分析在早期可以预测IVF和 SCNT胚胎。这些目标将提高农业和生物医学动物的产生效率模型并为人类临床应用建立策略和见解。