Development of efficient gene transfection procedure for animal cells

本研究利用阳离子脂质囊泡对动物细胞进行基因转移。为了提高转染效率，我们尝试了(1)添加DNA结合蛋白以保护DNA降解并促进核转移，(2)将配体引入脂质囊泡进行细胞特异性靶向，(3)与逆转录病毒整合酶结合以增强宿主基因组的整合。在DNA/阳离子脂质囊泡复合物形成之前，在DNA溶液中加入鱼精蛋白，可提高所有细胞系和所有血浆的转染效率和表达水平。与仅使用脂质囊泡相比，转染效率和表达水平的提高最多为20倍。我们还研究了用配体如胰岛素和半乳糖残基修饰脂质囊泡以实现受体介导的基因转移。通过胰岛素和半乳糖修饰的脂质囊泡，转染效率在所有细胞系和肝癌细胞系中分别提高了3-4倍。然后，我们试图将逆转录病毒整合机制纳入脂质囊泡介导的基因转染中，目的是实现高效稳定的转染。通过脂质囊泡介导的基因转染，用整合酶表达载体转染一个目的基因位于部分LTR序列之间的DNA片段。在最佳条件下，稳定转染效率比不加整合酶的转染效率提高了16倍。

We studied gene transfer for animal cells using cationic lipid vesicles. In order to enhance transfection efficiency, we attempted (1) addition of DNA binding proteins to protect DNA degradation and to promote nuclear transfer, (2) introduction of ligands to lipid vesicles for cell-specific targeting, and (3) combination with retrovirus integrase to enhance host genome in integration.By adding to protamine to DNA solution before the formation of DNA/cationic lipid vesicle complexes, transfection efficiency and expression level were enhanced for all the cell lines and all the plasma tested. The enhancement in both transfection efficiency and expression level was at most 20-fold compared with that using only lipid vesicles.We also examined modification of lipid vesicles with a ligand such as insulin and galactose-residues to realize receptor-mediated gene transfer. By insulin-and galactose-modified lipid vesicles, transfection efficiency increased 3-4 fold in all the cell lines tested and selectively in hepatoma cell lines, respectively.Then, we attempted to incorporate the retrovirus integration machinery in lipid vesicle-mediated gene transfection with the aim of achieving efficient stable transfection. A DNA fragment, in which a target gene was flanked between partial LTR sequences, was transfected with integrase expression vectors by means of lipid vesicle-mediated gene transfection. Under optimal conditions, the stable transfection efficiency showed a 16-fold improvement over that without integrase.