Functional Analysis of the Embryonic Epigenome in a Non-rodent Model

非啮齿动物模型中胚胎表观基因组的功能分析

• 批准号: 8063993
• 负责人: MARK E WESTHUSIN
• 金额: $ 28.13万
• 依托单位: TEXAS A&M AGRILIFE RESEARCH
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-15 至 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8063993
• 关键词: Acetylation; Address; Adult; Affect; Animal Model; Antibodies; Artificial Insemination; Assisted Reproductive Technology; Attention; Base Pairing; Biochemical; Birds; Breeding; Cardiovascular Diseases; Cardiovascular system; Cattle; Cell Count; Cell fusion; Cells; Chorion; Chromatin; Chromatin Structure Alteration; Code; Collection; Comparative Study; Conceptus; Cytosine; DNA; DNA Methylation; DNA Methyltransferase; DNA Modification Methylases; DNA Sequence; Development; Diabetes Mellitus; Diet and Nutrition; Disease; Elderly; Embryo; Embryo Transfer; Embryonic Development; Embryonic and Fetal Development; Endometrial; Environment; Enzymes; Epigenetic Process; Event; Failure; Female; Fertilization; Fertilization in Vitro; Fetal Development; Fetal Tissues; Future Generations; Gene Expression; Gene Expression Regulation; Gene Proteins; Gene Silencing; Gene Targeting; Genes; Genomics; Germ Cells; Goals; Gonadal structure; Guidelines; Health; Healthcare; Histology; Histones; Human; IGF Type 2 Receptor; Immune; In Vitro; Individual; Infertility; Investigation; K-18 conjugate; Laboratories; Life; Link; Malignant Neoplasms; Measures; Messenger RNA; Methods; Methylation; Methyltransferase Gene; Microarray Analysis; Modeling; Modification; Molecular; Molecular Analysis; Molecular Profiling; Monitor; Morula; Motion; Mus; Nature; Newborn Infant; Non-Rodent Model; Normalcy; Onset of illness; Oocytes; Oogenesis; Ovum; Pattern; Phosphorylation; Placentation; Play; Polymerase Chain Reaction; Pre-implantation Embryo Development; Pregnancy; Pregnancy Maintenance; Pregnancy Rate; Prevention; Procedures; Process; Production; Property; Protein Analysis; Proteins; Protocols documentation; Publications; RNA Interference; Receptor Gene; Regulation; Reporting; Reproduction; Reproductive Health; Research; Research Project Grants; Reverse Transcription; Rodent; Role; Small Interfering RNA; Spontaneous abortion; Staging; Staining method; Stains; Structure of placental cotyledon; Structure of primordial sex cell; Study models; Superovulation; Syndrome; System; Techniques; Testing; Time; Tissues; Toxic Environmental Substances; Transcript; Ultrasonography; Voice; Work; abortion; alcohol exposure; allantois; assisted reproduction; base; blastocyst; cell type; comparative; cost; demethylation; design; embryo cell; embryo culture; embryo stage 2; fetal; genome-wide; histone methyltransferase; histone modification; human disease; immunocytochemistry; implantation; imprint; improved; in vivo; insight; knock-down; novel therapeutic intervention; offspring; oocyte maturation; pluripotency; pregnant; preimplantation; programs; public health relevance; reproductive; research study; respiratory; response; trafficking; trophoblast; zygote

DESCRIPTION (provided by applicant): The long-term goal of the proposed research project is to characterize the factors and processes responsible for establishment of the mammalian epigenome, then decipher the mechanisms by which aberrations in epigenetic reprogramming which occur during preimplantation embryo development result in failed development or disease. Two key factors are associated with the epigenetic control of gene expression 1) DNA methylation and 2) histone modifications. Shortly after fertilization mammalian embryos undergo genome-wide epigenetic reprogramming by demethylation followed later by de novo remethylation. Aberrant epigenetic reprogramming has been clearly linked to failed embryo development and associated with serious human diseases. From the standpoint of human reproductive health and human disease, understanding the factors and mechanisms which control epigenetic reprogramming during early mammalian development is of critical importance. The objective of the proposed research is to generate scientific information that can be used to address these concerns. We propose to use a bovine model to pursue the following aims. Specific Aim 1 is to characterize the expression and activity of DNA methyltransferases (DNMTs), histone methyltransferases (HMTs) and their associated genes during mammalian oocyte maturation and pre-implantation development. Expression of genes coding for these enzymes in addition to protein quantity, localization and trafficking will be analyzed in bovine ova and embryos. Specific Aim 2 is to silence the expression of DNMTs and HMTs during bovine preimplantation development, then determine the effect on epigenetic reprogramming and embryo/fetal development. For study 1, RNA interference (RNAi) will be used transiently "knock down" expression of DNMTs and HMTs at the 1-cell stage of development. The percentage of embryos developing in vitro and embryo quality will be assessed. Global methylation in addition to methylation of specific target genes will also be analyzed. Microarray analysis and quantitative real-time PCR will be utilized to identify alterations in gene expression resulting from RNAi induced gene silencing. Immunocytochemistry will be employed to assess changes in protein. In a second study, siRNAs designed to silence the expression of DNMTs, HMTs and/or associated genes will be injected into one-cell bovine embryos which wil then be cultured to the blastocyst stage. These will be transferred into recipient females. Pregnancy rates will be compared between treatments and the normalcy of fetal development monitored by ultrasound. Conceptuses will be removed at 60 days of gestation to perform an extensive morphological and molecular analysis of placental and fetal tissues using methods similar to those described for study 1. Information generated in the proposed studies will be useful for generating guidelines and strategies to improve methods for assisted reproduction in humans, prevention of reproductive failure in addition to the prevention of human disease. PUBLIC HEALTH RELEVANCE: The long-term goal of the proposed research project is to characterize the factors and processes responsible for establishment of the mammalian epigenome, then decipher the mechanisms by which aberrations in epigenetic reprogramming which occur during embryo development result in failed development.The specific aims of this project involve the identification of key genes involved with epigenetic reprogramming during early mammalian development then employing RNAi based techniques to silence the expression of these genes and analyze the effects on embryo/fetal development.

描述（由申请人提供）：拟议的研究项目的长期目标是表征负责建立哺乳动物表观遗传组的因素和过程，然后破译了在植入前胚胎发育过程中发生表观遗传重编程的机制，导致发育或疾病失败。两个关键因素与基因表达的表观遗传控制有关1）DNA甲基化和2）组蛋白修饰。受精后不久，哺乳动物胚胎会通过脱甲基化进行全基因组表观遗传重编程，然后再进行从头二甲基化。异常表观遗传重编程显然与胚胎发育失败并与严重的人类疾病有关。从人类生殖健康和人类疾病的角度来看，了解控制早期哺乳动物发育过程中控制表观遗传重编程的因素和机制至关重要。拟议研究的目的是生成可用于解决这些问题的科学信息。我们建议使用牛模型来追求以下目标。具体目的1是在哺乳动物卵母细胞成熟和植入前发育过程中表征DNA甲基转移酶（DNMT），组蛋白甲基转移酶（HMT）及其相关基因的表达和活性。除蛋白质数量，定位和运输外，还将在牛OVA和胚胎中分析这些酶编码的基因表达。具体目标2是在牛植入前发育过程中静音DNMT和HMT的表达，然后确定对表观遗传重编程和胚胎/胎儿发育的影响。对于研究1，RNA干扰（RNAI）将在开发的1细胞阶段瞬时使用DNMT和HMT的表达。将评估在体外和胚胎质量发展的胚胎百分比。除特定靶基因的甲基化外，还将分析全球甲基化。微阵列分析和定量实时PCR将用于鉴定RNAi诱导的基因沉默引起的基因表达的改变。免疫细胞化学将用于评估蛋白质的变化。在第二项研究中，旨在使DNMT，HMTS和/或相关基因表达的siRNA被注入一细胞牛胚胎中，然后将其培养为胚泡阶段。这些将被转移到接受者的女性中。在治疗和通过超声监测的胎儿发育的正常水平之间将比较妊娠率。妊娠60天的概念将使用类似于研究1所描述的方法对胎盘和胎儿组织进行广泛的形态和分子分析。拟议的研究中产生的信息将有助于生成指南和策略，以改善人类辅助生殖的方法，预防人类病的再现失败。 PUBLIC HEALTH RELEVANCE: The long-term goal of the proposed research project is to characterize the factors and processes responsible for establishment of the mammalian epigenome, then decipher the mechanisms by which aberrations in epigenetic reprogramming which occur during embryo development result in failed development.The specific aims of this project involve the identification of key genes involved with epigenetic reprogramming during early mammalian development then employing RNAi based techniques to silence这些基因的表达并分析对胚胎/胎儿发育的影响。