Pharmaceutical studies for gene therapy: expression of human Cu, Zn-superoxide dismutase gene transfected by lipofection in rat skin fibroblasts.
To evaluate whether lipofection using Lipofectin is suitable for delivering foreign genes into skin fibroblasts as target cells, we performed experiments using human superoxide dismutase (hSOD) and neomycin-resistance (Neo) genes as models in rat skin fibroblasts (FR and primary cells) in vitro. The amounts of DNA used in the lipofection procedure significantly affected the transfection efficiencies, and the optimal amounts were determined for all cells used. However, the efficiencies in rat skin fibroblasts were about 20-fold higher than that in rat lung epithelial-like cells (L2 cells). The differences in plasmid vectors (pRc/RSV-SOD and pRc/CMV-SOD) hardly affected the transfection efficiencies. The amounts of Lipofectin significantly affected the transfection efficiencies, and the optimal amounts were determined for both types of skin fibroblasts. However, cytotoxic effects in both skin fibroblasts were observed with high doses of Lipofectin. On the other hand, with optimal amounts of DNA and Lipofectin, the reporter gene (NeoT) introduced into cells was mainly integrated into the host cell chromosome. Western blot analysis showed the continuous expression of hSOD protein for at least 45 d in skin fibroblasts transfected with the expression plasmid for hSOD by Lipofectin under the optimal conditions, and the cellular SOD activity fluctuated in parallel with the expression of hSOD protein. Differences in the type of cells also affected the expression of hSOD. These results indicate that it is necessary to set up optimal conditions for transfection using Lipofectin for each cell type, and that transfection with Lipofectin under optimal conditions may be an efficient method for introduction of foreign genes into skin fibroblasts for use as a clinical delivery system of therapeutic protein.
Nishiguchi
K
K
Department of Hospital Pharmacy, School of Medicine, Kobe University, Japan.
Ishida
K
K
Nakajima
M
M
Maeda
T
T
Komada
F
F
Iwakawa
S
S
Tanigawara
Y
Y
Okumura
K
K
D000818:Animals; D002478:Cells, Cultured; D005347:Fibroblasts / Q000378:metabolism; D015316:Genetic Therapy; D006801:Humans; D010714:Phosphatidylethanolamines / Q000494:pharmacology*; D051381:Rats; D013482:Superoxide Dismutase / Q000235:genetics*; D014162:Transfection
D010714:Phosphatidylethanolamines; C020888:1,2-dielaidoylphosphatidylethanolamine; D013482:Superoxide Dismutase