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缺陷型小鼠， 基因靶向 自申请人上次提交申请以来， 重要进展。 他创造了一种新的MTase敲除小鼠， 完全缺乏MTase活性。 此外，他还证明， 也是无效突变 两个突变等位基因 导致E10胚胎死亡。 正如李所承认的，这些MTase 有缺陷的小鼠可以回答许多重要的问题， 甲基化和印记：是否存在第二个MTase基因？MTase如何 有缺陷的胚胎存活下来，他们死亡的原因是什么？和 基因缺陷小鼠的基因表达如何受到影响？ 最近提出了两个根本性的重要意见。 第一 如上所述，创造了一种新的MTase缺陷小鼠， 线和示范，另一条线以前作出的事实上是 也有缺陷。 第二个观察结果是甲基化控制H19 以等位基因特异性方式表达。 建立了杂种ES细胞系 从MTase突变小鼠和野生型ES细胞来源于这些显示 H19的表达和甲基化是等位基因特异性的， 证明甲基化和印记在ES细胞中起作用， 甲基化是抑制父系等位基因所必需的。 现在提出实验来解决以下的序列特异性： 甲基化 最近两种不同印记基因的研究结果表明 这种去甲基化显示出序列特异性。 MTase缺陷小鼠可能 提供一种用于表征序列要求控制的系统， 甲基化的丧失。 具体目的1甲基化模式和基因表达的检查 包括DNA甲基化模式的表征 在突变胚胎和ES细胞中，并检查基因表达 模式. 待研究的基因包括Hbb、Pgk 2、Apoa 1、Alb、H19、Igf 2、 Snrnp、Xist、Pgk 1、Rps 4、Hprt等。 (The申请人最近 表明Xist，通常是父系印记， 在雄性突变胚胎中，认为甲基化是Xist所必需的， 镇压。） 具体目的2 MTase的组织和细胞缺陷的表征 突变体和MTase缺乏影响的方式的检查 细胞增殖和分化，包括组织学分析， 细胞增殖率、突变ES细胞的分化潜能 在体内和体外，和表征细胞死亡模式， MTase突变体。 还建议进行研究，以确定 细胞和组织缺陷，损害MTase缺陷的生存力 并检查MTase突变影响细胞的方式， 增殖和分化。 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查 和体内聚集嵌合体（使用lacZ标记），和 突变小鼠中细胞死亡性质的表征。 具体目的3确定甲基化， 染色质构象和印迹基因的表达，包括测试 对于印迹基因在MTase突变体中的表达，野生型- 型MTase在突变ES细胞中的表达及其对等位基因特异性的影响 H19基因的甲基化和表达，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.