Lipid-mediated transfection of normal adult human hepatocytes in primary culture.
The aim of this work was to develop a procedure for the lipid-mediated transfection of DNA into normal adult human hepatocytes in culture. Cells were plated in a serum-free culture medium at various cell densities, on plastic or collagen-coated dishes, both in the absence and in the presence of epidermal growth factor (EGF). The cells were incubated for various periods of time with mixtures of DNA-lipofectin or DNA-3 beta[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-chol) liposomes, and the efficiency of transfection was assessed by measuring the activity of reporter genes, beta-galactosidase or chloramphenicol acetyl-transferase (CAT). For comparison, similar experiments were carried out with human cell lines including HepG2, Caco-2, and WRL68. The efficiency of transfection (in percentage of cells) was not significantly different after transfection with lipofectin or DC-chol and comprised between 0.04 and 1.7% (extreme values) for different cultures. The efficiency of transfection decreased as the age or density of the culture increased and increased in cultures treated with EGF. Direct measurement of the rate of DNA synthesis suggested that the efficiency of transfection was related to the number of cells entering the S phase. Under the same conditions, the efficiency of transfection was one to two orders of magnitude greater in the three cell lines. A plasmid harboring 660 bp of the 5'-flanking region of CYP1A1 (containing two xenobiotic enhancer elements) fused upstream of the promoter of thymidine kinase and the CAT reporter gene was constructed. When this plasmid was transfected in human hepatocytes, CAT activity was induced as expected. We conclude that normal adult human hepatocytes can be transfected with exogenous DNA and that the transfected construct is regulated in the manner expected from in vivo studies.
Ourlin
J C
JC
INSERM U-128, CNRS, Montpellier, France.
Vilarem
M J
MJ
Daujat
M
M
Harricane
M C
MC
Domergue
J
J
Joyeux
H
H
Baulieux
J
J
Maurel
P
P
D000328:Adult; D002452:Cell Count; D002460:Cell Line; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D002784:Cholesterol / Q000031:analogs & derivatives; D019363:Cytochrome P-450 CYP1A1 / Q000096:biosynthesis / Q000235:genetics; D004247:DNA / Q000008:administration & dosage / Q000096:biosynthesis / Q000235:genetics; D004337:Drug Carriers; D004790:Enzyme Induction; D004815:Epidermal Growth Factor / Q000494:pharmacology; D017930:Genes, Reporter; D006801:Humans; D008055:Lipids; D008081:Liposomes; D008099:Liver / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D008854:Microscopy, Electron; D010714:Phosphatidylethanolamines; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000235:genetics
D004337:Drug Carriers; D008055:Lipids; D008081:Liposomes; D010714:Phosphatidylethanolamines; C070648:3-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol; D004815:Epidermal Growth Factor; C020888:1,2-dielaidoylphosphatidylethanolamine; D004247:DNA; D002784:Cholesterol; D019363:Cytochrome P-450 CYP1A1; D015500:Chloramphenicol O-Acetyltransferase; D001616:beta-Galactosidase