MOUSE MODELS OF HUMAN PWS/AS IMPRINTING CENTER MUTATIONS

哺乳动物中的大多数基因在母体和母体中均等表达 父系遗传的等位基因。然而，有些基因是带有印记的， 仅由一个亲本等位基因表达。印记的后果之一 是表达的等位基因中的单个突变或缺失，将 尽管存在野生型，但结果是缺乏基因产物 型，但沉默等位基因。这是涉及的突变类型 遗传病普瑞德威利综合症（PWS）和天使综合症（AS）， 代表由相反的结果产生的不同的临床表型 人类染色体 15q11-q13 的基因组印记模式。的 本提案中描述的实验将牢固地确立小鼠 作为人类 15q11-q13 印记的模型系统并开始解决 更复杂的机械问题可能无法解决 人类。 第一个具体目标是创建人类 PWS 和 AS 的小鼠模型 印记突变来检验上游区域的假设 Snrpn 基因是一个关键的印记中心，负责 多个印记基因的全局调控。 如果我们找到证据 有了印记中心，我们就发挥我们的能力 在亲本上创建独立但相同的缺失突变 小鼠等位基因研究印记中心的机制 以及非重叠 AS 和 PWS 删除的相关性。的 第二个具体目标是确定两个 Snrpn 中的任何一个是否打开 阅读框架在印记过程中发挥作用并评估每个 开放阅读框架对 PWS 的贡献。第三个具体目标是 重点关注 Snrpn 位点及其周围的印迹机制 确定如何分配父母身份的方法。这将涉及到 确定是否存在亲本特异性甲基化差异以及是否 附近有任何转录单位。 第四个具体目标是 开发一种功能测定法来鉴定顺式作用元件 导致 Snrpn 基因被印记，并随后确定是否 最小的鉴定序列足以赋予父本特异性 表达到另一个基因。

Most genes in mammals are expressed equally from the maternally and paternally inherited alleles. However, some genes are imprinted, and are expressed from only one parental allele. One consequence of imprinting is that a single mutation or deletion in the expressed allele, will result in the absence of a gene product despite the presence of a wild- type, but silent allele. This is the type of mutation involved in the genetic diseases Prader-Willi Syndrome (PWS) and Angelman Syndrome (AS), which represent distinct clinical phenotypes resulting from opposite patterns of genomic imprinting of human chromosome 15q11-q13. The experiments described in this proposal will firmly establish the mouse as a model system for imprinting of human 15q11-q13 and begin to address the more intricate mechanistic questions that may be unapproachable in humans. The first specific aim is to create mouse models of human PWS and AS imprinting mutations to test the hypothesis that the region upstream of the Snrpn gene serves as a critical imprinting center, responsible for the global regulation of multiple imprinted genes. If we find evidence that there is an imprinting center, we will take advantage of our ability to create independent but identical deletion mutations on both parental alleles in mice to investigate the mechanism of the imprinting center as well as the relevance of the non-overlapping AS and PWS deletions. The second specific aim is to determine if either of the two Snrpn open reading frames play a role in the imprinting process and evaluate each open reading frame's contribution to PWS. The third specific aim will focus on the imprinting machinery in and around the Snrpn locus as a means of determining how parental identity is assigned. This will involve determining if there is parent-specific methylation differences and if there are any nearby transcriptional units. The fourth specific aim is to develop a functional assay to identify the cis-acting elements that cause the Snrpn gene to be imprinted and subsequently determine if the minimal identified sequence are sufficient to confer paternal-specific expression to another gene.