Epigenetic Variation in Mice with Defective Imprinting

描述（由申请人提供）：本研究的主要目标是了解在缺乏Dnmt1胞嘧啶甲基转移酶dnmt10形式的小鼠胚胎发育中异常表型的原因。要测试的中心前提是，大多数缺乏dnmt10的胚胎死亡是因为它们是具有缺陷印记的细胞的表观遗传嵌合体。因此，它们不能正常发育。该模型系统将允许详细检查表观遗传变异对表型和妊娠结局的影响。目的1：确定来自缺乏dnmt10的卵母细胞的胚胎的表观遗传嵌合体的性质。来自Dnmt1（delta10）纯合子雌性的胚胎在缺乏母体提供的dnmt10的情况下发育，并表现出50%的正常甲基化等位基因的DNA甲基化缺失。每个胚胎都是一个嵌合体，应该由许多不同的表观遗传细胞类型组成，从印迹基因上大多数正常甲基化的细胞到印迹基因上几乎没有甲基化的细胞。这一预测将通过测量来自dnmt10缺陷胚胎的胚胎干（ES）和胚胎成纤维细胞（EF）克隆的印迹甲基化模式来验证。目的2：建立dnmt10缺陷卵母细胞胚胎中表观遗传嵌合体的来源。dnmt10依赖性维持甲基化的缺失可能发生在胚胎的第四个S期。这一假设将通过测量每个植入前阶段的dnmt10缺陷胚胎池中的印迹甲基化模式，以及使用每个dnmt10缺陷植入前阶段的供体细胞核衍生的克隆胚胎来验证。目的3：确定dnmt10缺陷胚胎的可变异常表型的原因。缺乏dnmt10的胚胎会死亡，很可能是因为它们的印迹缺陷细胞无法促进正常发育。这一假设将通过首先定义dnmt10缺陷胚胎和胚胎外组织中存在的形态学异常来验证。随后，将测试dnmt10缺陷胚胎干细胞的克隆（每个克隆都具有独特的正常和缺陷印记模式）对ES囊胚和ES四倍体嵌合体胚胎发育的贡献能力。

DESCRIPTION (provided by applicant): The primary goal of this research is to understand the cause of the abnormal phenotypes in mouse embryos developing in the absence of the Dnmt1o form of the Dnmt1 cytosine methyltransferase. The central premise to be tested is that the majority of Dnmt1o-deficient embryos die because they are epigenetic mosaics of cells that have defective imprints. Consequently, they do not develop normally. This model system will allow a detailed examination of the effect of epigenetic variation on phenotype and pregnancy outcome. Aim 1: Determine the nature of the epigenetic mosaicism in embryos derived from Dnmt1o-deficient oocytes. An embryo derived from a Dnmt1(delta1o) homozygous female develops in the absence of maternally supplied Dnmt1o and exhibits a loss of DNA methylation from 50% of the normally methylated alleles of imprinted genes. Each embryo is a mosaic, and should be comprised of many different epigenetic cell types, ranging from cells with mostly normal methylation on imprinted genes to cells with nearly no methylation on imprinted genes. This prediction will be tested by measuring imprinted methylation patterns in clones of embryonic stem (ES) and embryonic fibroblast (EF) cells derived from Dnmt1o-deficient embryos. Aim 2: Establish the source of the epigenetic mosaicism in embryos derived from Dnmt1o-deficient oocytes. The loss of Dnmt1o-dependent maintenance methylation probably occurs at the fourth embryonic S phase. This hypothesis will be tested by measuring imprinted methylation patterns in pools of Dnmt1o-deficient embryos at each preimplantation stage, and in cloned embryos derived using donor nuclei from each Dnmt1o-deficient preimplantation stage. Aim 3: Determine the cause of the variable, abnormal phenotypes of Dnmt1o-deficient embryos. Dnmt1o-deficient embryos die, most likely because their imprint-defective cells are unable to contribute to normal development. This hypothesis will be tested by first defining the morphological abnormalities present in Dnmt1o-deficient embryos and extraembryonic tissues. Afterwards, clones of Dnmt1o-deficient ES cells, each a self-renewing population with a unique pattern of normal and defective imprints, will be tested for their ability to contribute to embryonic development in ES-blastocyst and ES-tetraploid chimeras.