| Budget Amount *help |
¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 1999: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1998: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
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.
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