Studies on the β-galactosidosis using transgenic mice expressing ganglioside-related glycogenes

使用表达神经节苷脂相关糖原的转基因小鼠研究β-半乳糖苷沉积症

• 批准号: 13680918
• 负责人: MATSUDA Junichiro
• 金额: $ 2.24万
• 依托单位: National Institute of Infectious Diseases
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13680918/
• 关键词: Mouse; Disease model; Transgenic; Lysosomal disease; β-Galactosidase; β-Galactosidosis; Gangliosidosis; Neurodegenerative disease

β-Galactosidosis comprises clinically different diseases, GM1-gangliosidosis and Morquio B disease, both of which are caused by deficiency of lysosomal acid β-galactosidase (β-Gal). We focused on GM1-gangliosidosis, a neurogenetic disease occurring mainly in early infancy, and rarely in childhood or young adults. In this study, we generated the model mouse of juvenile GM1-gangliosidosis, expressing a mutant enzyme protein R201C on the β-Gal KO background. The model mice exhibited slowly progressive neurological deterioration, such as tremor and gait disturbance from 9 months of age and died around 15 months. Storage of ganglioside GM1 in their brains was less abundant than in the β-Gal KO mouse brain. Using this mouse, we examined the possibility of chemical chaperone therapy. Oral administration of newly synthesized β-Gal inhibitor to the juvenile GM1-gangliosidosis model mice resulted in significant enhancement of the enzyme activity in the brain and other tissues. Although mass biochemical analysis did not show the reduction of GM1 in the brain in this short term trial (1 week), chemical chaperone therapy may be useful for certain patients with GM1-gangliosidosis and potentially other lysosomal storage diseases with central nervous system involvement. We also generated the transgenic mice expressing GM1/GA1 synthase gene and tried to introduce the transgene into β-Gal KO mice in order to accelerate their disease onset. We injected the transgene fragments (3.5 kb) containing CAG promoter and mouse GM1/GA1 synthase cDNA (1.2 kb) into fertilized oocytes of C57BL/6J mice. We produced 4 lines of transgenic mice expressing the transgene at least in the liver and the brain. GM1 was detected in the liver of 3 transgenic mouse lines, although no GM1 was detectable in non-transgenic C57BL/6 mice. We are currently evaluating the effect of the over expression of GM1/GA1 synthase on the disease onset by introducing the transgene into β-Gal KO mice.

β-半乳糖苷脂沉积症包括临床上不同的疾病，GM 1-神经节苷脂沉积症和Morquio B病，这两种疾病都是由溶酶体酸性β-半乳糖苷酶（β-Gal）缺乏引起的。我们集中在GM 1-神经节苷脂沉积症，主要发生在婴儿早期的神经遗传性疾病，很少在儿童或年轻人。在这项研究中，我们产生了幼年型GM 1-神经节苷脂沉积症的模型小鼠，在β-Gal KO背景下表达突变酶蛋白R201 C。模型小鼠从9个月大开始表现出缓慢进行性的神经功能恶化，例如震颤和步态障碍，并在15个月左右死亡。神经节苷脂GM 1在它们脑中的储存量低于β-Gal KO小鼠脑中的储存量。使用这种小鼠，我们研究了化学伴侣治疗的可能性。对幼年GM 1-神经节苷脂沉积症模型小鼠口服新合成的β-Gal抑制剂导致脑和其他组织中的酶活性显著增强。尽管大量的生化分析没有显示在这个短期试验（1周）中大脑中GM 1的减少，但化学伴侣治疗可能对某些患有GM 1-神经节苷脂沉积症和其他可能累及中枢神经系统的溶酶体贮积病的患者有用。我们还建立了转GM 1/GA 1合成酶基因小鼠，并尝试将转基因导入β-Gal KO小鼠，以加速其发病。将含有CAG启动子和GM 1/GA 1合成酶cDNA的转基因片段（3.5kb）注射到C57 BL/6 J小鼠受精卵中。我们产生了至少在肝脏和大脑中表达转基因的4系转基因小鼠。在3个转基因小鼠品系的肝脏中检测到GM 1，但在非转基因C57 BL/6小鼠中未检测到GM 1。我们目前正在通过将转基因导入β-Gal KO小鼠来评估GM 1/GA 1合酶过表达对疾病发作的影响。

项目成果

Noguchi A et al.: "Chromosomal mapping and zygosity check of transgenes based on flanking genome sequences determined by genomic walking."Exp Anim. Vol.53. 103-111 (2004)

Noguchi A 等人：“基于基因组行走确定的侧翼基因组序列，对转基因进行染色体作图和接合性检查。”Exp Anim。

Itoh M et al.: "Developmental pathology of mice with targeted disruption of the β-galactosidase gene : a model of human G_<M1>-gangliosidosis."Brain and Development. 23. 379-384 (2001)

Itoh M 等人：“β-半乳糖苷酶基因靶向破坏的小鼠的发育病理学：人类 G_<M1>-神经节苷脂沉积症模型。”《大脑与发育》23. 379-384 (2001)。

Noguchi A et al.: "Chromosomal mapping and zygosity check of transgenes based on flanking genome sequences determined by genomic walking."Exp Anim. 53. 103-111 (2004)

Noguchi A 等人：“基于基因组行走确定的侧翼基因组序列，对转基因进行染色体作图和接合性检查。”Exp Anim。

Yamamoto Y et al.: "Phenotype correction of GM1-gangliosidosis mice by the introduction of a human β-galactosidase transgene (in Japanese with an English abstract)."Proceedings of the Japanese Society of Animal Models for Human Diseases. 17. 20-22 (2001)

Yamamoto Y 等人：“通过引入人类 β-半乳糖苷酶转基因来校正 GM1 神经节苷脂沉积症小鼠的表型（日文版，附英文摘要）。”日本人类疾病动物模型学会会议记录 17. 20-。 22 (2001)

Tominaga, L et al.: "Galactonojirimycin derivatives restore mutant human p-galactosidase activities expressed in fibroblasts from enzyme-deficient knockout mouse."Brain and Development. Vol.23. 284-287 (2001)

Tominaga, L 等人：“半乳糖野尻霉素衍生物可恢复酶缺陷型敲除小鼠成纤维细胞中表达的突变型人类β-半乳糖苷酶活性。”《大脑与发育》。