Electroporation-mediated gene transfer into hepatocytes: preservation of a growth hormone response.
An electroporation procedure is described which allows the introduction of foreign genes into freshly isolated rat hepatocytes while preserving their growth hormone responsiveness. A single-pulse procedure performed at low voltage (150-200 V) but with high capacitance (960 microF), conditions which caused minimal cell damage and increased hepatocyte survival in culture (greater than 80%), was found to be optimal for both the basal and the hormone-stimulated expression of transfected genes. Transfection of the cells suspended in a phosphate buffer at high concentrations (20-25 x 10(6)/ml) with large amounts of plasmid (30 micrograms/assay) gave the best results. Raising the temperature up to 25 or 37 degrees C (instead of 4 degrees C) decreased about twofold basal CAT expression but appeared to increase the magnitude (i.e., fold induction) of hormonal effects. Expression of the reporter gene driven by either a viral or a liver gene promoter reached a maximum after 24 h, a situation especially favorable when studying liver-specific gene expression known to decay rapidly in cultured hepatocytes. This procedure was successfully applied to the study of a growth hormone-dependent serine protease inhibitor gene promoter.
Paquereau
L
L
Centre de Pharmacologie-Endocrinologie, Centre National de la Recherche Scientifique, Montpellier, France.
Le Cam
A
A
D000818:Animals; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004560:Electricity; D015870:Gene Expression / Q000187:drug effects; D005821:Genetic Techniques; D013006:Growth Hormone / Q000494:pharmacology*; D066298:In Vitro Techniques; D008099:Liver / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D011401:Promoter Regions, Genetic; D051381:Rats; D015842:Serine Proteinase Inhibitors / Q000235:genetics; D014162:Transfection
D015842:Serine Proteinase Inhibitors; D013006:Growth Hormone; D015500:Chloramphenicol O-Acetyltransferase