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、无价的人类遗传病动物模型，不适合在啮齿动物身上进行研究。这种潜力还没有完全实现，因为产生零突变的技术还没有应用到猴子身上。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。绕过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，这个过程应该会导致零突变动物的发育。卡尔曼综合症是一种人类疾病的一个特别突出的例子，在这种疾病中，零突变猴子可以提供重要的新信息。Kallmann综合征患者表现出两个严重的异常：1)他们缺乏嗅球，导致嗅觉障碍；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基因的功能突变引起的。目前，还没有这种疾病的功能动物模型。我们提出了三个具体的目标：1)确定KAL-1mRNA在恒河猴中的时空表达；2)构建KAL-1基因打靶载体；3)干扰KAL-1在恒河猴细胞中的表达。这三个目标的成功完成将为一项通过将KAL-1基因被破坏的细胞的细胞核转移到去核卵母细胞来培育KAL-1缺失突变猕猴的提议提供初步数据。这种动物模型的发展是合理的，原因有三。首先，尚未鉴定出KAL-1的小鼠同源基因。其次，在人类中，这种基因的缺失不会导致胚胎死亡，但确实会导致一种在新生儿身上可以通过非侵入性手段观察到的表型。第三，KAL-1基因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这种基因的一个拷贝，就可以完全消除第一只产生的动物中KAL-1的表达。如果这个项目的长期目标得以实现，就会产生一个无价的卡尔曼综合症模型。此外，还将建立一种使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.