MOUSE MODELS OF HUMAN PWS/AS IMPRINTING CENTER MUTATIONS

哺乳动物中的大多数基因在母体和 父系遗传的等位基因然而，有些基因是印记的， 仅由一个亲本等位基因表达。印记的一个后果是 在表达的等位基因中的单个突变或缺失， 导致基因产物的缺失，尽管存在野生型- 型，但沉默等位基因。这是一种突变类型， 遗传疾病Prader-Willi综合征（PWS）和Angelman综合征（AS）， 它们代表了由相反的 人类染色体15 q11-q13的基因组印记模式。的 本提案中描述的实验将牢固地建立小鼠 作为人类15 q11-q13印迹的模型系统，开始解决 更复杂的机械问题，可能是无法接近的， 人类 第一个具体目标是建立人类PWS和AS的小鼠模型 印记突变来检验上游区域的假设 Snrpn基因作为一个关键的印记中心，负责 多个印记基因的全局调节。 如果我们找到证据 有一个印记中心，我们将利用我们的能力 在两个亲本上产生独立但相同的缺失突变， 等位基因在小鼠研究机制的印记中心， 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中的任一个是否打开 阅读框在印迹过程中发挥作用，并评估每个 开放式阅读框架对PWS的贡献。第三个具体目标是 专注于Snrpn基因座内和周围的印记机制， 确定父母身份如何分配的手段。这将涉及 确定是否存在亲本特异性甲基化差异， 附近是否有转录单位 第四个具体目标是 开发一种功能性测定方法，以鉴定顺式作用元件， 导致Snrpn基因被印记，并随后确定 最小识别序列足以赋予父系特异性 另一个基因的表达。

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.