Transfection of hepatic genes into adult rat hepatocytes in primary culture and their tissue-specific expression.
We describe in this paper a method for studying transient gene expression in a primary culture of adult rat hepatocytes. After isolation by collagenase perfusion, hepatocytes in a monolayer were transfected with foreign DNA by the calcium phosphate precipitation technique during the first 24 hours after plating. When they were transfected with a plasmid containing the gene for chloramphenicol acetyltransferase driven by the early promoter of simian virus 40, hepatocytes reproducibly expressed high levels of chloramphenicol acetyltransferase (CAT); this transient expression was much higher than that obtained with the rat hepatoma cell line H4II. Different medium conditions have been tested; an optimal level of CAT activity can be obtained using a serum-free, hormonally defined medium. Using these techniques, we have investigated the expression of liver-specific genes transferred into hepatocytes. We show that the L-pyruvate kinase promoter is active in these hepatocytes while it is silent in fibroblasts. Moreover, the use of serum-free medium may allow investigation of the role of hormones and nutrients in cells which respond normally to these effectors.
Ginot
F
F
Unité de Recherches en Génétique et Pathologie Moléculaires, Unité 129 de l'Institut National de la Santé et de la Recherche Médicale, Paris, France.
Decaux
J F
JF
Cognet
M
M
Berbar
T
T
Levrat
F
F
Kahn
A
A
Weber
A
A
D000818:Animals; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics* / Q000378:metabolism; D005796:Genes; D005798:Genes, Bacterial; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D010957:Plasmids; D051381:Rats; D011919:Rats, Inbred Strains; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D011994:Recombinant Proteins; D015500:Chloramphenicol O-Acetyltransferase