Application of HVJ liposome for creating the next generation of animal models for human diseases.

应用 HVJ 脂质体创建下一代人类疾病动物模型。

• 批准号: 10680776
• 负责人: KITADA Kazuhiro
• 金额: $ 2.11万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10680776/
• 关键词: HVJ liposome; transfection into live animals; animal models for human diseases; gene therapy; mutamt; positinal cloning; ALY mouse; tremor rat; トランスジェニック動物; LacZ

1. Establishment of transfection methods into live animals by HVJ liposomeWe constructed two expression vectors, pEBActNII-LacZ and pEBActNII-GFP, and generated HVJ liposome from each of them. 50 microL of HVJ liposomes were injected into cerebral cortex of neonates(1 or 3 days of age), and the animals were examined by LacZ staining or under fluorescence microscope. As a result, we detected expression of these genes in the applied portions of the central nervous system.2. Trial of the transgenesis by injection of HVJ liposome into testesWe injected HVJ liposome containing pEBActNII-LacZ into testes, and obtained offsprings from the manipulated animals for examining the rate of transgenesis. However, no germ-line transmission was observed in 84 animals in our research. This finding suggested that the rate of transgenesis by injection of HVJ liposome into testes is too low to generate transgenic animals efficiently.3. Positional cloning of the causative genes in animal models for human diseasesThe tremor rat is a mutant that exhibits absence-like seizure and spongiform degeneration in the central nervous system (CNS). By positional cloning, a genomic deletion was found within the critical region , in which the aspartoacylase gene is located. Accordingly, no aspartoacylase expression was detected in any of the tissues examined, and abnormal accumulation of N-acetyl-L-aspartate (NAA) was shown in the mutant brain, in correlation with the severity of the vacuole formation. Therefore, the tremor rat may be regarded as a suitable animal model of human Canavan disease, characterized by spongy leukodystrophy that is caused by aspartoacylase deficiency.

1.HVJ脂质体转染活体动物方法的建立我们构建了pEBActNII-LacZ和pEBActNII-GFP两种表达载体，并从它们中分别产生了HVJ脂质体。将50μL HVJ脂质体注射到新生儿（1或3日龄）的大脑皮质中，并进行LacZ染色或荧光显微镜下检查。结果，我们检测到这些基因在中枢神经系统应用部位的表达。2．睾丸注射HVJ脂质体转基因试验我们将含有pEBActNII-LacZ的HVJ脂质体注射到睾丸中，并从操作动物中获得后代用于检测转基因率。然而，在我们的研究中，84 只动物中没有观察到生殖系传播。这一发现表明，睾丸注射HVJ脂质体的转基因率太低，无法有效产生转基因动物。 3．人类疾病动物模型中致病基因的定位克隆震颤大鼠是一种突变体，在中枢神经系统（CNS）中表现出失神样癫痫发作和海绵状变性。通过定位克隆，在天冬氨酸酰化酶基因所在的关键区域内发现了基因组缺失。因此，在任何检查的组织中都没有检测到天冬氨酸酰化酶的表达，并且在突变体大脑中显示出N-乙酰基-L-天冬氨酸（NAA）的异常积累，这与液泡形成的严重程度相关。因此，震颤大鼠可被视为人类卡纳万病的合适动物模型，其特征是由天冬氨酸酰化酶缺乏引起的海绵状脑白质营养不良。

项目成果

Shinkura, R.et al: "Alymphoplasia is caused by a point mutation in the mouse gene encoding Nf-kappa b-inducing kinase."Nature Genetics. 22. 74-77 (1999)

Shinkura, R.等人：“淋巴发育不全是由编码 Nf-kappa b 诱导激酶的小鼠基因中的点突变引起的。”《自然遗传学》。

Kitada,K.et al.: "Positional cloning of the causative gene for a neurological mutant rat tremor : Identification of the genomic deletion causing aspartoacylase deficiency"Jounal of Neurochemistry. (in press).

Kitada，K.等人：“神经突变大鼠震颤致病基因的定位克隆：导致天冬氨酸酰化酶缺乏的基因组缺失的鉴定”神经化学杂志。

Saeki,Y.et al.: "Sustained transgene expression in vitro and in vivo using an Epstein-Barr virus replicon vector system combined with HVJ-liposomes"Gene Thrapy. 5. 1031-1037 (1998)

Saeki, Y. 等人：“使用 Epstein-Barr 病毒复制子载体系统与 HVJ 脂质体相结合，在体外和体内持续进行转基因表达”基因治疗。

Hagihara,Y.et al.: "Wide spread gene transfection into the central nervous system in rats"Gene Therapy. (in press).

Hagihara, Y. 等人：“将基因广泛转染至大鼠中枢神经系统”基因治疗。

Saeki,Y.et al.: "Sustained transgene expression in vitro and in vivo using an Epstein-Barr virus replicon vector system combimed with HVJ-liposomes"Gene Therapy. 5. 1031-1037 (1998)

Saeki, Y. 等人：“使用 Epstein-Barr 病毒复制子载体系统与 HVJ 脂质体相结合，在体外和体内持续进行转基因表达”基因治疗。