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

1. 构建pEBActNII-LacZ和pEBActNII-GFP两个表达载体，分别生成HVJ脂质体。将50 μ l HVJ脂质体注射于1日龄或3日龄新生儿大脑皮层，采用LacZ染色或荧光显微镜观察。结果，我们在中枢神经系统的应用部分检测到这些基因的表达。将含pebactni - lacz的HVJ脂质体注射入睾丸，获得经处理动物的后代，观察其转基因率。然而，在我们的研究中84只动物未观察到种系传播。这一发现表明，通过注射HVJ脂质体进入睾丸的转基因率太低，无法有效地产生转基因动物。人类疾病动物模型中致病基因的位置克隆震颤大鼠是一种突变体，在中枢神经系统（CNS）中表现出缺失样癫痫和海绵状变性。通过定位克隆，在天冬氨酸酰化酶基因所在的关键区域发现了基因组缺失。因此，在检查的任何组织中均未检测到天冬氨酸酰化酶的表达，并且在突变的大脑中显示n -乙酰- l-天冬氨酸（NAA）的异常积累，这与液泡形成的严重程度有关。因此，震颤大鼠可作为人类卡纳万病的合适动物模型，卡纳万病以天门冬氨酸酰化酶缺乏引起海绵状脑白质营养不良为特征。

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.