CHROMOSOMAL SITE FOR KRABBE'S DISEASE GENE

鞘脂病是一组糖脂遗传性疾病 代谢，由于溶酶体酶缺乏，可导致精神 发育迟缓，往往是致命的。 目前对这些疾病的研究 包括更深入地了解其精确的遗传基础， 它们在基因组中的定位以及它们的分子生物学。 的 酶促真实小鼠模型的可用性（抽搐小鼠） 克拉伯病是一种半乳糖神经酰胺β-半乳糖苷酶缺乏症， 人类，极大地促进了对这些疾病的基础研究，并提供了 用于测试基因和酶替代疗法的动物系统。 这项研究使用了半乳糖苷酶缺陷的ticker小鼠 在人类基因组中定位这种酶的结构基因， 体细胞遗传学技术 来自3天皮肤样品的细胞系 已经建立了新生抽搐小鼠，并在 这个实验室一年半（超过200个细胞倍增）。 这些永久性的半乳糖苷酶缺陷系将与 显示半乳糖苷酶活性的正常人二倍体细胞， 用携带显性基因的逆转录病毒使新霉素耐药， 这种特质。 将在新霉素（G418）培养基中选择杂交细胞， 存在将消除两种亲本细胞类型的胰蛋白酶。 的 然后克隆杂交体，并测定单个克隆的 半乳糖苷酶活性和人同工酶含量。 单身的人类 一组半乳糖脑苷酶阳性克隆共有的同工酶标记 将为该基因的染色体定位提供第一个证据 因为任何半乳糖苷酶 这些杂交细胞中的活性必然来自人类 基因 所提出的策略是独特的，并且避免了使用免疫学或 区分人与鼠的理论方法 人类X小鼠杂交体中的酶。 这被认为是一个非常可取的 在这些研究中使用的抽搐小鼠的特征，因为酶 半乳糖苷酶特别难以纯化用于可靠的纯化。 抗体的产生，并且也不能用 电泳研究的组织化学方法。 今后的工作 这些细胞将包括使用染色体的基因区域定位， 显带技术和基因分离研究。

The sphingolipidoses are a group of genetic disorders of glycolipid metabolism, due to lysosomal enzyme deficiencies, that can result in mental retardation and are often fatal. Current studies of these diseases involves a deeper understanding of their precise genetic basis, including their localization within the genome and their molecular biology. The availability of an enzymatically authentic mouse model (the twitcher mouse) for Krabbe's disease, a galactosylceramide beta-galactosidase deficiency in humans, greatly facilitates basic studies on these disorders and provides an animal system for testing gene and enzyme replacement therapies. The proposed studies use the galactocerebrosidase-deficient twitcher mouse to localize the structural gene for this enzyme in the human genome using somatic cell genetics techniques. Cell lines from skin samples of 3 day neonate twitcher mice have been established and continuously propagated in this laboratory for one and one-half years (over 200 cell doublings). These permanent galactocerebrosidase-deficient lines will be crossed with normal human diploid cells exhibiting galactocerebrosidase activity and made neomycin resistant with a retrovirus carrying the dominant gene for this trait. Hybrid cells will be selected in neomycin (G418) medium in the presence of oubain which will eliminate both parental cell types. The hybrids will then be cloned and individual clones assayed for galactocerebrosidase activity and human isozyme contents. The single human isozyme marker shared by a panel of galactocerebrosidase-positive clones will provide the first evidence for the chromosmal localization of the gene for this enzyme in the human genome, since any galactocerebrosidase activity in these hybrid cells must necessarily have come from the human gene. The proposed strategy is unique and avoids the use of immunological or electrophoretical methods for distinguishing the human from the mouse enzyme in human X mouse hybrids. This considered an extremely desirable feature of the twitcher mouse for use in these studies, since the enzyme galactocerebrosidase has been particularly difficult to purify for reliable antibody production and also, cannot be distinguished specifically using the histochemical methods for electrophoresis studies. Future work with these cells will include regional mapping of the gene using chromosmal banding techniques and gene isolation studies.