Introduction of the foreign genetic information into tissue cells of living animals and its application to the medical science.

在本项目期间，我们取得了以下成果。1.利用多层膜融合脂质体将基因导入动物组织我们通过将脂质体与仙台病毒孵育并在蔗糖梯度上纯化来制备包封DNA和蛋白质的多层膜融合脂质体。该脂质体可将大肠杆菌β-半乳糖苷酶基因和人B型肝炎病毒表面抗原基因导入大鼠肝细胞并表达。2. Singlelamella融合脂质体的纯化及性质研究我们通过反相法制备脂质体，与仙台病毒共同孵育，然后在蔗糖梯度上纯化，制备了包裹DNA和蛋白质的Singlelamella融合脂质体。我们发现，这些脂质体基本上不含非融合脂质体和病毒，并且它们具有更高的将大分子引入细胞的效率（<10>每10D_1单位的蛋白质为10^7个颗粒，DNA为10^7个颗粒<540>）。这些纯化的脂质体将其内容物引入细胞质的效率与完整仙台病毒的效率相同。我们还发现这些脂质体的直径为250 nm。3.仙台病毒突变株持续感染的机理分析我们证明了ts突变株Cl 151的持续感染是由于其突变M蛋白在非允许温度下不稳定。我们还表明，M蛋白不影响病毒的基因表达。这是开发基于仙台病毒的病毒载体的第一步。4.人类人工染色体载体的研制成功地构建了人类人工染色体。载体（pMYAC 1、pMYAC 2）由人端粒、作为酵母人工染色体维持的酵母DNA、作为人细胞筛选标记的耐药基因组成。这是构建人类人工染色体的第一步，人类人工染色体是人类基因治疗的最终系统。

We obtained the following results during the term of this project. 1.Gene Introduction into Animal Tissues Using Multilamella Fusogenic Liposomes.We prepared multilamella fusogenic liposomes encapsulated DNA and protein by incubating liposomes with Sendai virus and purifying them on sucrose gradient. These liposomes could introduce and express E.coli beta-galactosidase gene and the surface antigen gene of human hepatitis B virus into liver cells of living rats. 2.Purification and Characterization of Singlelamella Fusogenic Liposomes.We prepared singlelamella fusogenic liposomes encapsulating DNA and protein by preparing liposomes by reverse phase method, incubating them with Sendai virus and purifying them on sucrose gradient. We found that these liposomes were essentially free from unfusogenic liposomes and virus and that they had higher efficiency of introduction of macromolecules into cells(10^<10> particles for protein and 10^7 particles for DNA per 1 OD_<540> unit). The efficiency of these purified liposomes to introduce their contents into cytoplasm was the same as that of intact Sendai virus. We also found that these liposomes had a diameter of 250nm. 3.Analysis of Mechanism of Persistent Infection of Mutant Sendai Virus.We demonstrated the ts mutant, Cl151, Caused persistent infection because their mutant M protein was unstable at non-permissive temperature. We also showed that M protein did not affect the gene expression of the virus. This is a first step for the development of the virus vector based on Sendai virus. 4.Development of the Vectors for Human Artificial Chromosome.We succeeded to make the vectors construct the human artificial chromosome. The vectors(pMYAC1,pMYAC2) consisted of human telomere, yeast DNA for maintaining as yeast artificial chromosome, drug resistant genes as selection markers in human cells. This is the first step for construction of human artificial chromosome, an ultimate system for human gene therapy.