DNA METHYLATION IN GENOMIC IMPRINTING AND DEVELOPMENT

描述：此修订后的应用程序的目标是使用 转基因小鼠研究基因突变的分子基础 印记和印记不平衡的发展后果。 这些实验是基于MTase缺陷小鼠，通过 基因打靶。自上次提交以来，申请人已提交 取得了重要进展。他创造了一种新的MTase基因敲除小鼠 谷氨酸转氨酶活性完全不足。此外，他还证明了淘汰赛 他前段时间所做的也是一个零突变。两个突变的等位基因 导致胚胎在10岁时死亡。正如李所认识到的，这些MTase 有缺陷的小鼠使回答许多重要的问题成为可能 甲基化和印记：是否存在第二个MTase基因？MTase是如何 缺陷胚胎存活下来，它们的死亡原因是什么？ 缺陷小鼠的基因表达是如何受到影响的？ 最近出现了两个重要的观察结果。第一 如上所述，创造了一种新的MTase缺陷小鼠 以及之前所做的另一条线的演示实际上是 也有缺陷。第二个观察结果是甲基化控制了H19 以等位基因特有的方式表达。建立了杂交系ES细胞。 来自MTase突变小鼠和来自这些显示的野生型ES细胞 H19的表达和甲基化是等位基因特异性的，因此 证明甲基化和印记在胚胎干细胞中起作用 这种甲基化是抑制父亲的等位基因所必需的。 现在建议进行实验来解决序列特异性问题 甲基化。最近对两种不同印记基因的研究结果表明 这种去甲基化表现出序列特异性。MTase缺陷小鼠可能 提供一种用于表征序列需求控制的系统 甲基化的丧失。 特定目标1甲基化模式和基因表达的检查 在MTase突变中包括DNA甲基化模式的特征 在突变的胚胎和ES细胞中，以及基因表达的检测 模式。待研究的基因包括Hbb、Pgk2、ApoA1、Alb、H19、Igf2、 SnRNP、Xist、Pgk1、RPS4、HPRT等。(申请人最近 显示了Xist，通常是父系印记的，表达 在男性突变胚胎中，认为Xist需要甲基化 压抑。) 特异靶2 MTase组织和细胞缺陷的特征 MTase缺乏症的突变体和影响方式的检测 细胞增殖和分化，包括组织学分析， 突变型ES细胞的细胞增殖率和分化潜能 在体内和体外，以及细胞死亡模式的表征 MTase突变体。还建议进行研究，以确定 影响MTase缺陷患者生存能力的细胞和组织缺陷 并检测MTase突变对细胞的影响 增殖分化。这些研究将涉及 突变胚胎的组织学分析、细胞增殖试验 ES细胞体外分化潜能的检测 和体内聚合嵌合体(使用LacZ标记)，以及 突变小鼠细胞死亡性质的表征。 具体目标3确定甲基化、 印迹基因的染色质构象和表达，包括检测 对于MTase突变体中印记基因的表达，野生型- 突变ES细胞的MTase类型及其对等位基因特异性的影响 H19基因甲基化和表达，以及H19基因的检测 野生型和mtase突变体中h19基因的染色质结构。

DESCRIPTION: The goals of this revised application are to use genetically altered mice to investigate the molecular basis for imprinting and the developmental consequences of imprinting imbalances. These experiments are based on MTase deficient mice produced through gene targeting. Since the previous submission the applicant has made important progress. He has created a new MTase knock-out mouse that is fully deficient in MTase activity. Moreover he showed that the knock-out that he made some time ago is also a null mutation. Both mutant alleles result in embryonic lethality at E10. As Li recognizes, these MTase deficient mice make it possible to answer many important questions about methylation and imprinting: Is there a second MTase gene?, How do MTase deficient embryos survive, and what is the cause of their death?, and How is gene expression affected in deficient mice? Two fundamentally important observations were recently made. The first was, as mentioned above, the creation of a new MTase deficient mouse line and the demonstration that another line made previously is in fact also deficient. The second observation is that methylation controls H19 expression in an allele-specific manner. A hybrid ES cell line was made from MTase mutant mice and wild-type ES cells derived from these show that expression and methylation of H19 is allele-specific, thereby demonstrating that methylation and imprinting operate in ES cells and that methylation is required for repression of the paternal allele. Experiments are now proposed to address the sequence specificity of methylation. Recent results for two different imprinted genes suggest that demethylation shows sequence specificity. MTase deficient mice may provide a system for characterizing the sequence requirements controlling the loss of methylation. Specific Aim 1 Examination of methylation patterns and gene expression in MTase mutants including characterization of DNA methylation patterns in mutant embryos and ES cells, and examination of gene expression patterns. Genes to be studied include Hbb, Pgk2, Apoa1, Alb, H19, Igf2, Snrnp, Xist, Pgk1, Rps4, Hprt and others. (The applicant recently showed that Xist, which is normally paternally imprinted, is expressed in male mutant embryos, arguing that methylation is required for Xist repression.) Specific Aim 2 Characterization of tissue and cellular defects of MTase mutants and examination of the manner in which MTase deficiency affects cell proliferation and differentiation, including histological analyses, cell proliferation rates, differentiation potential of mutant ES cells in vivo and in vitro, and characterization of cell death patterns in MTase mutants. Studies are also proposed to determine the nature of the cellular and tissue defect that compromises viability in MTase deficient embryos and to examine the way in which the MTase mutation affects cell proliferation and differentiation. These studies will involve histological analyses of mutant embryos, tests of cell proliferation rates, examination of differentiation potential for ES cells in vitro and in vivo in aggregation chimeras (using a lacZ marker), and characterization of the nature of cell death in mutant mice. Specific Aim 3 Determination of the relationship between methylation, chromatin conformation and expression of imprinted genes, including tests for expression of imprinted genes in MTase mutants, expression of wild- type MTase in mutant ES cells and its effects on allele-specific methylation and expression of the H19 gene, and examination of the chromatin structure of the H19 gene in wild-type and MTase mutants.