Higher efficiency of retrovirus transduction in the late stage of primary culture of hepatocytes from nontreated than from partially hepatectomized rat.
The aim of this investigation was to optimize the conditions for gene transduction by a retroviral vector into primary cultured hepatocytes. Because a retrovirus infection is dependent upon the proliferative activity of the target cells, we examined the thymidine incorporation of the primary culture, using cells from nontreated and partially hepatectomized donor rats. Partial hepatectomy 1 day before cell isolation greatly enhanced the thymidine incorporation, and similarly, the transduction of LacZ, the gene for E. coli beta-galactosidase, although the percentage of transduced cells remained low. With cells from the normal rat, the thymidine incorporation increased gradually after the beginning of the culture and reached 16 times the initial activity on day 2, while hepatocytes from the partially hepatectomized donor showed no increase at that stage of the culture. Correspondingly, the number of gene-transduced hepatocytes was increased when the vector was added to the cells 1 or 2 days after isolation. Therefore, hepatocytes cultured for 1 or 2 days after isolation are suitable for retroviral transduction.
Department of Experimental Surgery and Bioengineering, National Children's Medical Research Center, Tokyo, Japan.
S
S
Enosawa
S
S
Suzuki
X K
XK
Li
T
T
Okuyama
M
M
Fujino
H
H
Amemiya
D000818:Animals; D017690:Cell Transplantation / Q000379:methods*; D002478:Cells, Cultured; D005822:Genetic Vectors; D006498:Hepatectomy; D007763:Lac Operon / Q000235:genetics; D008099:Liver / Q000166:cytology*; D008297:Male; D051381:Rats; D011916:Rats, Inbred F344; D012190:Retroviridae; D013936:Thymidine / Q000378:metabolism; D014162:Transfection / Q000379:methods*
D013936:Thymidine