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

点击翻译按钮获取中文摘要

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.