| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1996: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1995: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
We investigated cationic liposome-mediated DNA transfection in the presence of serum.
Transfection efficiency decreased dramatically with an increasing of serum concentration and the efficiency in medium containing 5% calf serum was nearly half of that in serum-free medium. An addition of albumin to medium also lowered the efficiency and to 35% at a concentration of 50 mg/ml. DNA stability and the effect of incubation times were further studied to elucidate the effect of serum on transfection. Analysis of DNA degradation with agarose electrophoresis showed that DNA complexed with cationic liposomes were more resistant to nuclease in serum than free plasmid DNA.Furthermore, in the presence of serum, transfection efficiency increased over time. The efficiency at 30 hour was 3-fold higher than that at 3 hour, and it was almost comparable to that in normal transfection in serum-free medium. The longer incubation was achieved by reducing the cytotoxicity of DNA-liposome complexes in medium c
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ontaining serum. These results suggested that lowered transfection efficiency in the presence of serum is mainly due to a delay in the uptake process of DNA-liposome complexes in transfection, rather than an irreversible change in DNA.
The transfection ability and cytotoxicity of the synthetic polypeptides and the effect of molecular weight and concentration of the polypeptide on DNA transfection were examined.
Poly (L-ornithine) , poly (L-arginine) and poly (L-ornithine, L-leucine) induced very high expression levels of a foreign gene in HeLa S3 cells. However, poly (L-lysine) and poly (L-lysine, L-alanine) were not effective. Cytotoxicity was observed at high concentrations of polypeptides and increased in the order poly (L-ornithine) >poly (L-lysine) >poly (L-arginine) <greater than or equal>poly (L-ornithine, L-leucine) >poly (L-lysine, L-alanine) . The molecular weight of polypeptide also greatly influenced on the transfection activity. The higher molecular weight of poly (L-ornithine) (MW203,400) mediated genc transfer most effectively, and its transfection activity were approximately 5-fold higher than that of poly (L-ornithine) with a molecular weight of 53,600, whereas the poly (L-ornithine) of a lower molecular weight (MW11,700) exhibited little activity. The optimal concentrations of the polypeptides depended on the DNA level in an applied solution. The optimal mass ratio of DNA/polypeptide for transfection was 0.4-0.8. The transfection activity of a polypeptide depends on its amino acid composition , molecular weight and DNA/peptide ratio. High molecular poly (L-ornithine) and poly (L-arginine) are more efficient mediators of DNA transfection than Lipofectin, a commercially available Less
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