Improving Bovine Cloning Efficiency by Enhancing Reprogramming during Embryonic Genome Activation (EGA)

这个F32提案概述了研究目标，以调查早期基因表达调控 牛体细胞核移植（SCNT）克隆胚胎的发育 通过利用我们对IVF牛植入前发育的知识，基于几 发表的报告，包括我自己的工作，我的假设是，失败的胚胎基因组激活， (EGA)是对克隆动物的健壮发育产生负面影响的限速步骤。 由于许多EGA激活的基因参与了胚胎染色质的表观遗传重编程， 不能适当地打开这些基因导致发育失调和失败， 通常在妊娠后期表现出来。 基于我们已发表的工作，表明DUX转录因子（TF）是主要的 调节基因表达在鸡蛋到胚胎过渡，并利用我广泛的 未发表的初步数据奠定了基础工作，我们的假设在牛的系统，我将 首先使用尖端的基因组学、表观基因组学和胚胎学方法来解决这个问题。 首先，我将测试我在体外受精牛胚胎中发现的假定增强子区域是否显示出 体内阶段特异性转录活性。我也将剖析哪些TF的假定 增强子区域响应于使用细胞系统。然后我将识别开放染色质 区域，并将其与高质量的IVF胚胎开放染色质图谱进行比较 我以前创造的。这将帮助我了解SCNT的发展 表型是由于不能激活EGA染色质或不能使体细胞退役而引起的 开放染色质。最后，我将使用我们的数据，牛DUXC是牛EGA的主要激活剂， 检验我们假设，DUXC缺陷可以通过在SCNT中异位表达DUXC来克服 这将提高SCNT的效率。对人类辅助的意义 生殖技术，人类胚胎发育的大型动物模型的开发，农业 克隆的意义，以及通过SCNT保护濒危物种的生态影响， 这项工作整合了影响多个领域的创新战略。

This F32 proposal outlines research objectives to investigate the early gene expression regulation of bovine somatic cell nuclear transfer (SCNT) cloned embryos, and improve their developmental potential by harnessing our knowledge of IVF bovine preimplantation development. Based on several published reports, including my own work, my hypothesis is that failed embryonic genome activation (EGA) is the rate-limiting step negatively impacting the robust development of cloned animals. Because many EGA activated genes are involved in epigenetic reprogramming embryonic chromatin, failure to appropriately turn on these genes leads to developmental dysregulation and failure, often manifested much later in gestation. Building upon both our published work, showing that DUX transcription factors (TF) are the master regulators of gene expression at the egg-to-embryo transition, and utilizing my extensive unpublished preliminary data laying the ground work for our hypothesis in the bovine system, I will first address this problem using cutting-edge genomic, epigenomic, and embryological methods. First, I will test whether the putative enhancer regions I identified in IVF bovine embryos show stage-specific transcriptional activity in vivo. I will also dissect which TFs the putative enhancer regions are responsive to using a cellular system. Then I will identify open chromatin regions in bovine SCNT embryos and compare them to the high-quality IVF embryo open chromatin maps I have previously generated. This will help me understand how much of the SCNT developmental phenotype is caused by failure to activate EGA chromatin or failure to decommission somatic cell open chromatin. Finally, I will use our data that cow DUXC is a major activator of bovine EGA to test our hypothesis that DUXC deficiency can be overcome by ectopically expressing DUXC in SCNT embryos and that this will increase the efficiency of SCNT. With significance for human assisted reproductive technologies, development of large animal models of human embryogenesis, agricultural significance of cloning, and ecological impacts of endangered species preservation through SCNT, this work integrates innovative strategies to impact multiple fields.