Altered Nuclear Transfer

改变核转移

• 批准号: 8013894
• 负责人: SHOUKHRAT M MITALIPOV
• 金额: $ 53.81万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-01 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8013894
• 关键词: Autologous; CDX2 gene; Cell Line; Cell Nucleus; Cells; Degenerative Disorder; Derivation procedure; Development; E-Cadherin; Embryo; Epigenetic Process; Inner Cell Mass; Maintenance; Mediating; Modeling; Molecular Profiling; Monkeys; Mus; Oocytes; Outcome; Patients; Pluripotent Stem Cells; Production; Role; Small Interfering RNA; Somatic Cell; Stem cells; Testing; Therapeutic; Up-Regulation; blastocyst; improved; insight; mouse model; nonhuman primate; nuclear transfer; pre-clinical; prevent; public health relevance; research study; somatic cell nuclear transfer; transcription factor

DESCRIPTION (provided by applicant): The aim of this project is to generate important new insights concerning epigenetic factors in oocytes governing reprogramming following somatic cell nuclear transfer (SCNT) and formation and maintenance of the first two lineages in mammalian embryos, the inner cell mass (ICM) and trophectoderm (TE). Our main hypothesis is that experimental modulation of expression levels for the key transcription factors in oocytes will prevent formation of the TE while enhancing the production of the ICM lineage. These alterations should preclude the formation of an embryo while promoting and enhancing donor nucleus reprogramming required for the formation of a single lineage from which pluripotent stem cell lines can be established. We propose the following specific aims: Aim 1. Establish a mouse model of ANT with improved ESC derivation. Initially, we will investigate the effect of siRNA-mediated knockdown of Cdx2, Tead4, and E-Cadherin in mouse oocytes on reprogramming of somatic cells after SCNT. Next, we will examine a cumulative impact of the TE inactivation along with upregulation of critical pluripotent factors - Oct4, Sox2, and Nanog - on reprogramming and derivation of ESCs. Aim 2. Define factors in monkey oocytes that are crucial for TE development. In Experiment 1, we will examine the presence and expression profiles of TEAD4 in monkey oocytes and preimplantation embryos. Next, we will study the role of TEAD4 in monkey oocytes and embryos by morpholino-assisted knockdown. Lastly, fertilized TEAD4-deficient oocytes will be used for ESC isolation. Aim 3: Analyze the roles of key maternal TE factors in epigenetic reprogramming after monkey SCNT. In this aim, we will apply most promising approaches to assess the validity of ANT in the monkey model by first creating CDX2 and TEAD4 depleted oocytes and then conducting SCNT and subsequently deriving ESCs from the resultant pluripotent cells. 1 PUBLIC HEALTH RELEVANCE: In this application we will study the potential of autologous (patient-matched) stem cells derived by somatic cell nuclear transfer in the nonhuman primate model while avoiding the destruction of viable embryos. The outcomes of these experiments, in turn, will allow the production of useful preclinical monkey models for testing therapeutic applications involving autologous stem cells for the treatment of wide range of degenerative diseases.

描述（由申请人提供）：该项目的目的是在体细胞核转移（SCNT）（SCNT）以及在哺乳动物胚胎中的前两个谱系，内部细胞质量（ICM）和滋养型（Te）中产生有关重新编程的卵母细胞中的重要新见解。我们的主要假设是，卵母细胞中关键转录因子的表达水平的实验调节将阻止TE形成TE，同时增强ICM谱系的产生。这些改变应排除胚胎的形成，同时促进和增强形成单个谱系所需的供体核重编程，从而从中建立了多能干细胞系。我们提出以下特定目的：目标1。建立具有改进的ESC推导的ANT小鼠模型。最初，我们将研究siRNA介导的CDX2，TEAD4和E-钙粘着蛋白在小鼠卵母细胞中的敲低对SCNT后体细胞重编程的影响。接下来，我们将研究TE失活的累积影响以及关键多能因素的上调OCT4，SOX2和NANOG对ESC的重编程和衍生。 AIM 2。定义对TE发育至关重要的猴子卵母细胞中的因素。在实验1中，我们将检查猴子卵母细胞和植入前胚胎中TEAD4的存在和表达谱。接下来，我们将研究Tead4在猴子卵母细胞中的作用，并通过形态辅助敲低的胚细胞和胚胎。最后，受精的TEAD4缺陷型卵母细胞将用于ESC隔离。 AIM 3：分析关键母体TE因子在猴子SCNT后表观遗传重编程中的作用。在此目标中，我们将采用最有前途的方法来评估猴子模型中ANT的有效性，首先创建CDX2和TEAD4耗尽的卵母细胞，然后进行SCNT，然后进行SCNT，然后从所得的多能细胞中得出ESC。 1 公共卫生相关性：在本应用中，我们将研究由非人类灵长类动物模型中体细胞核转移得出的自体（患者匹配）干细胞的潜力，同时避免破坏可行的胚胎。这些实验的结果反过来将允许生产有用的临床前猴模型，用于测试涉及自体干细胞的治疗应用，以治疗多种退行性疾病。