Culture time-dependent gene expression in isolated primary cultured rat hepatocytes by transfection with the cationic liposomal vector TFL-3.
The development of a carrier system that enables the transfer of a functional exogenous gene to non- or less frequently dividing mammalian cells is essential for increasing the available options for the treatment of various diseases. The issue of whether TFL-3, a recently developed cationic liposome, can be successfully used to achieve gene expression in primary cultured rat hepatocytes was examined. The hepatocytes were transfected for 4 h with plasmid DNA (pDNA) in TFL-3 at various time points after 4-h preculture. The transfection efficiency was determined at various times posttransfection with pDNA coding for chloramphenicol acetyltransferase (CAT), luciferase, or beta-galactosidase. The amount of intranuclear pDNA present, as a consequence of the lipofection, was also quantitatively determined. Successful lipofections were observed for all pDNA tested, and the efficiencies were superior to that of commercially available LIPOFECTAMINE under our experimental conditions. The degree and rate of gene expression were dependent on incubation time prior to lipofection as well as on the density of the cells per dish, but this relationship did not hold for the amount of gene delivered to the nuclei. These results indicate that TFL-3 could be a useful vector for achieving sufficient gene expression in rat hepatocytes and suggest that the culture time prior to and following lipofection, which is related to the biological condition of the cells, may be one major factor affecting efficient gene expression in nondividing cells.
Nguyen
Lap Thi
LT
Department of Pharmacokinetics and Biopharmaceutics, Faculty of Pharmaceutical Sciences, The University of Tokushima, 1-78-1 Sho-machi, Tokushima 770-8505, Japan.
Ishida
Tatsuhiro
T
Ukitsu
Sachiko
S
Li
Wen Hao
WH
Tachibana
Rieko
R
Kiwada
Hiroshi
H
D000818:Animals; D002412:Cations; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D022781:Hepatocytes / Q000378:metabolism*; D008081:Liposomes; D008297:Male; D010957:Plasmids / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics
D002412:Cations; D008081:Liposomes; D015500:Chloramphenicol O-Acetyltransferase; D001616:beta-Galactosidase