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A RHESUS MACAQU MODEL OF KALLMANN'S SYNDROME

卡尔曼综合征的恒河猴模型

基本信息

批准号：
6188463
负责人：
ROBERT B. NORGREN
金额：
$ 14.33万
依托单位：
UNIVERSITY OF NEBRASKA MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-09-30 至 2002-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6188463
关键词：
Kallmann's syndrome Macaca mulatta disease /disorder model gene expression gene targeting genetic library genetic models immunocytochemistry in situ hybridization model design /development nuclear transfer sex chromosomes sex linked trait transfection /expression vector

项目摘要

Rhesus macaques closely resemble humans; as such they represent a potentially invaluable animal model for human genetic diseases not amenable to study in rodents. This potential has not been fully realized as the technology for generating null mutants has not been applied to monkeys. The fact that macaques require 4 years before they are able to breed has been a key impediment to the generation of null mutants. One way to circumvent this problem is to disrupt genes in cells and then transfer the nuclei of these cells to enucleated oocytes. If male cells are used, and the gene is on the X chromosome, this procedure should result in the development of a null mutant animal. Kallmann's syndrome is a particularly salient example of a human disease in which null mutant monkeys could provide important new information. Patients with Kallmann's syndrome exhibit two serious abnormalities: 1) they lack olfactory bulbs resulting in anosmia and 2) they do not have LHRH neurons in their brains resulting in hypogonadism. These symptoms are caused by loss of function mutations in the KAL-1 gene. Currently, there is no functional animal model of this disease. We propose three specific aims in this project: 1) to determine the spatial-temporal expression of KAL-1 mRNA in the rhesus macaque; 2) to construct a KAL-1 gene targeting vector; 3) to disrupt KAL-1 in rhesus macaque cells. Successful completion of these three aims would provide preliminary data for a proposal to produce a KAL-1 null mutant rhesus macaque by transferring the nuclei from cells with disrupted KAL-1 genes to enucleated oocytes. The development of this animal model is justified for three reasons. First, a mouse homolog of KAL-1 has not been identified. Second, deletion of this gene in humans is not embryonic lethal but does result in a phenotype that is observable in newborns by noninvasive means. Third, KAL-1 is located on the X chromosome in humans and rhesus macaques. Therefore, it is only necessary to disrupt one copy of this gene, in male cells, to completely abolish KAL-1 expression in the first animal produced. If the long term goals of this project are accomplished, an invaluable model of Kallmann's syndrome would be generated. In addition, a powerful new methodology for the study of human diseases using the rhesus macaque would be established.

恒河猴与人类非常相似;因此，它们代表了不适合在啮齿动物中研究的人类遗传疾病的潜在无价动物模型。由于产生无效突变体的技术尚未应用于猴子，这种潜力尚未完全实现。事实上，猕猴需要4年才能繁殖，这是产生无效突变体的关键障碍。解决这个问题的一种方法是破坏细胞中的基因，然后将这些细胞的细胞核转移到去核的卵母细胞中。如果使用雄性细胞，并且基因位于X染色体上，则该程序应导致无效突变动物的发育。卡尔曼综合征是人类疾病的一个特别突出的例子，其中无效突变的猴子可以提供重要的新信息。卡尔曼综合征患者表现出两种严重的异常：1）他们缺乏嗅球，导致嗅觉丧失，2）他们的大脑中没有LHRH神经元，导致性腺功能减退。这些症状是由KAL-1基因的功能缺失突变引起的。目前，还没有这种疾病的功能性动物模型。本课题的主要目的是：1）研究恒河猴KAL-1 mRNA的时空表达规律; 2）构建KAL-1基因打靶载体; 3）在恒河猴细胞中破坏KAL-1基因。这三个目标的成功完成将为通过将细胞核从具有破坏的KAL-1基因的细胞转移到去核卵母细胞来产生KAL-1无效突变恒河猴的提议提供初步数据。该动物模型的开发基于三个原因。首先，尚未鉴定出KAL-1的小鼠同源物。第二，人类中该基因的缺失不是胚胎致死性的，但确实会导致通过非侵入性手段在新生儿中观察到的表型。第三，KAL-1位于人类和恒河猴的X染色体上。因此，只需要在雄性细胞中破坏该基因的一个拷贝，就可以完全消除所产生的第一只动物中的KAL-1表达。如果这个项目的长期目标得以实现，将产生一个宝贵的卡尔曼综合征模型。此外，还将建立一种利用恒河猴研究人类疾病的强有力的新方法。