Development of efficient gene transfection procedure for animal cells

我们研究了利用阳离子脂泡进行动物细胞的基因转移。为了提高转染率，我们尝试了(1)添加DNA结合蛋白来保护DNA降解和促进核移植，(2)在脂泡上引入配体以实现细胞特异性靶向，(3)结合逆转录病毒整合酶来增强宿主基因组的整合。通过在DNA/阳离子脂泡复合体形成之前向DNA溶液中加入鱼精蛋白，所有细胞系和所有受试血浆的转染率和表达水平都得到了提高。与单纯使用脂泡相比，转基因效率和表达水平最多提高了20倍。我们还研究了用胰岛素和半乳糖残基等配体修饰脂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泡，在所有受试细胞系和肝癌细胞系中的转染率分别提高了3-4倍。然后，我们尝试将逆转录病毒整合机制整合到脂泡介导的基因转染法中，以实现高效稳定的转染率。通过脂泡介导的基因转染法，将目的基因夹在部分LTR序列之间的DNA片段与整合酶表达载体共转染。在最佳条件下，稳定的转染率比没有整合酶时提高了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.