CHROMOSOMAL SITE FOR KRABBE'S DISEASE GENE

鞘磷脂病是一组糖脂遗传性疾病 由于溶酶体酶缺乏而导致的新陈代谢，可导致精神疾病 发育迟缓，往往是致命的。关于这些疾病的最新研究 涉及到对它们确切的遗传基础的更深入的理解，包括 它们在基因组中的定位及其分子生物学。这个 一种酶活性可靠的小鼠模型(抽动小鼠)的可用性 对于Krabbe病，半乳糖基神经酰胺β-半乳糖苷酶缺乏症 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。 拟议的研究使用了半乳脑苷酶缺陷的抽动小鼠 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。取自3日龄皮肤样本的细胞系 已经建立了新生的抽动小鼠，并在 这个实验室一年半了(超过200个细胞加倍)。 这些永久性的半乳脑苷酶缺陷型品系将与 显示半乳糖脑苷酶活性的正常人二倍体细胞和 使新霉素与携带显性基因的逆转录病毒产生抗药性 这一特点。杂交细胞将在新霉素(G418)培养基中选择。 欧巴因的存在将消除两种亲本细胞类型。这个 杂交种将被克隆，并对单个克隆进行检测 半乳糖脑苷酶活性和人体同工酶含量。单身的人类 一组半乳糖脑苷酶阳性克隆共享的同工酶标记 将为基因的染色体定位提供第一个证据 对于人类基因组中的这种酶，因为任何半乳脑苷酶 这些杂交细胞中的活动一定来自人类 吉恩。 建议的策略是独一无二的，避免使用免疫学或 区分人和小鼠的电泳法 人X鼠杂交种中的酶。这被认为是非常可取的 在这些研究中使用的抽动鼠的特征，因为酶 半乳脑苷酶一直是特别难纯化的可靠的 抗体的产生，而且，不能特别区分使用 电泳法研究的组织化学方法。未来的工作与 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.