EARLY MAMMALIAN DEVELOPMENT AND DNA METHYLATION

描述（改编自研究者摘要）：这是一个新的 一位有前途的年轻研究员的申请， DNA甲基化在早期基因表达控制中的作用 发展 有四个具体目标。 第一，检查 甲基化对几种病毒启动子功能的详细影响。 通过使用聪明的体外甲基化策略和染色体外EBV 基于载体系统，PI将询问独特位点中的甲基化是否 在测试的启动子内或在转录区中的突变可以影响表达。 在第二个具体目标中，蛋白质介导的DNA环将在 通过将lac操纵子位点策略性地置于其它基于EBV的质粒内， 测试质粒并将这些质粒引入哺乳动物细胞， 支持EBV载体的复制，并组成型表达 lacI基因 通过使用适当甲基化的底物， 可以探测环状和非环状（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珠蛋白基因区的不同部分的甲基化， 含有β珠蛋白基因座的P1克隆上的甲基化模式 并将其导入胚胎干细胞， 转化成可以表达珠蛋白基因的细胞。 第四个具体目标是 尝试通过分离含有印迹的P1克隆来研究印迹 H19和IGF2基因，并以各种方式将它们甲基化， 植入胚胎干细胞来测试对印记的影响。

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.