EARLY MAMMALIAN DEVELOPMENT AND DNA METHYLATION

描述(改编自《调查者摘要》)：这是一个新的 来自一位有前途的年轻研究员的申请，他的目的是研究各种 DNA甲基化在早期基因表达调控中的作用 发展。有四个具体目标。第一个是检查 甲基化对几种病毒启动子功能的详细影响。 通过使用巧妙的体外甲基化策略和染色体外EBV 基于载体系统，PI会询问甲基化是否在唯一的位点 在被测试的启动子内或在转录区域可以影响表达。 在第二个具体目标中，蛋白质介导的DNA环将在 通过策略性地将lac操纵子位点放置在 测试质粒并将这些质粒导入哺乳动物细胞，这将 支持EBV载体的复制并构成表达 Laci基因。通过使用适当的甲基化底物，稳定性 通过使用IPTG，可以探测到环和非环(通过使用IPTG)质粒中的甲基化。 第三个具体目标是调查特定地点的作用 通过改变β珠蛋白基因区域不同部分的甲基化 含有β珠蛋白基因的P1克隆的甲基化模式 并将它们导入ES细胞，然后使其分化 转化成可以表达珠蛋白基因的细胞。第四个具体目标是 试图通过分离包含印记的P1克隆来研究印记 H19和IGF2基因，以不同的方式甲基化并重新引入 转化为ES细胞，以测试其对印迹的影响。

DESCRIPTION (Adapted from the Investigator's Abstract): This is a new application from a promising young investigator designed to study a variety of aspects of DNA methylation in the control of gene expression in early development. There are four specific aims. The first is to examine the detailed effects of methylation on the function of several viral promoters. By using a clever in vitro methylation strategy and an extrachromosomal EBV based vector system, the PI will ask whether methylation in unique sites within the tested promoters or in transcribed regions can affect expression. In the second specific aim, protein mediated DNA looping will be created in other EBV based plasmids by placing lac operator sites strategically within test plasmids and introducing these plasmids into mammalian cells which will support the replication of the EBV vectors and which constitutively express the lacI gene. By using suitably methylated substrates, the stability of methylation in looped and unlooped (by using IPTG) plasmids can be probed. The third specific aim is to investigate the role of site specific methylation of various portions of the beta globin gene region by altering the methylation patterns on P1 clones which contain the beta globin locus and introducing them into ES cells which can then be made to differentiate into cells which can express globin genes. The fourth specific aim is to try to study imprinting by isolating a P1 clone which contains the imprinted H19 and IGF2 genes and methylate them in various ways and reintroduce them into ES cells to test the effect on imprinting.