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的表达是一样的 父系遗传的等位基因。然而，一些基因是被印记的，而且是 仅表达于一个亲本等位基因。印记的一个后果 表达的等位基因中的一个突变或缺失，就会 导致基因产物的缺失，尽管存在野生的- 类型，但沉默的等位基因。这是一种突变类型，涉及到 遗传病Prader-Willi综合征(PWS)和Angelman综合征(AS)， 它们代表了截然不同的临床表型 人类染色体15q11-q13的基因组印迹模式。这个 这项提案中描述的实验将牢牢地建立起老鼠 作为人类15q11-q13印记的模型系统，并开始解决 更复杂的机械性问题，可能是 人类。 第一个具体目标是建立人类PWS和AS的小鼠模型 印记突变以检验假设，即 Snrpn基因是一个关键的印迹中心，负责 多个印记基因的全球调控。如果我们找到证据 有一个印记中心，我们会利用我们的能力 在两个亲本上创建独立但相同的缺失突变 小鼠的等位基因来研究印记中心AS的机制 以及不重叠的AS和PWS删除的相关性。这个 第二个特定目标是确定两个Snrpn中的任何一个是否打开 阅读框架在印记过程中起着作用，并对每个框架进行评估 开放阅读框架对PWS的贡献。第三个具体目标是 重点关注Snrpn轨迹内和周围的印记机制 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 附近有没有任何转录单位。第四个具体目标是 开发一种功能分析来鉴定顺式作用元件 使Snrpn基因被印记，然后确定是否 最小已识别序列足以赋予父系特异性 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.