A RHESUS MACAQU MODEL OF KALLMANN'S SYNDROME

恒河猴与人类非常相似；因此，它们代表了一种潜在的宝贵的动物模型，用于研究无法在啮齿动物身上进行研究的人类遗传疾病。由于产生零突变体的技术尚未应用于猴子，因此这种潜力尚未完全实现。猕猴需要4年才能繁殖，这一事实一直是零突变体产生的关键障碍。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，并且基因在X染色体上，这个过程应该导致零突变动物的发展。卡尔曼氏综合征是人类疾病的一个特别突出的例子，无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，导致嗅觉缺失；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基因的功能突变丧失引起的。目前，尚无功能性动物模型。我们提出了本项目的三个具体目标：1)确定恒河猴中KAL-1 mRNA的时空表达；2)构建KAL-1基因靶向载体；3)破坏恒河猴细胞中的KAL-1。这三个目标的成功完成将为通过将KAL-1基因中断的细胞的细胞核转移到去核卵母细胞中来产生KAL-1零突变恒河猴的提议提供初步数据。这种动物模型的发展有三个理由。首先，KAL-1的小鼠同源物尚未确定。其次，在人类中，这种基因的缺失并不会导致胚胎死亡，但确实会导致一种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，就可以在第一只动物中完全消除KAL-1的表达。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。

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.