Retrovirus-mediated transduction of adult hepatocytes.
Retrovirus-mediated gene transfer was used to develop a method for introducing genes into primary cultures of adult rat hepatocytes. Subconfluent monolayers of hepatocytes, cultured in hormonally defined media on different matrix substrata, were infected with helper-free stocks of a replication-defective retrovirus that constitutively expresses high levels of beta-galactosidase. Retrovirus-mediated transduction was measured by two methods: (i) an in situ cytochemical stain that specifically detects the expression of viral expressed beta-galactosidase, and (ii) Southern blot analysis, which measures the relative copy number of integrated provirus. Maximal transduction efficiency of approximately equal to 25% was achieved when the cells were infected after 3 days in culture; matrix had little effect on transduction efficiency. Enzyme cytochemical (catalase and glucose 6-phosphatase) and peroxidase immunocytochemical (asialoglycoprotein and UDP-glucuronosyltransferase) analyses of the cultures indicated that greater than 95% of cells were hepatocytes. The demonstration of hepatocyte-specific organelles in cells expressing the viral-directed beta-galactosidase provided unambiguous evidence for the transduction of hepatocytes. These methods should be useful in the development of liver-directed somatic gene therapy and in the study of liver-specific gene regulation.
Wilson
J M
JM
Whitehead Institute for Biomedical Research, Cambridge, MA 02142.
Jefferson
D M
DM
Chowdhury
J R
JR
Novikoff
P M
PM
Johnston
D E
DE
Mulligan
R C
RC
D000818:Animals; D002460:Cell Line; D005109:Extracellular Matrix / Q000032:analysis; D005814:Genes, Viral; D005952:Glucose-6-Phosphatase / Q000032:analysis; D014453:Glucuronosyltransferase / Q000032:analysis; D006651:Histocytochemistry; D008099:Liver / Q000648:ultrastructure*; D051379:Mice; D008830:Microbodies / Q000201:enzymology; D051381:Rats; D012190:Retroviridae / Q000235:genetics*; D014162:Transfection; D001616:beta-Galactosidase / Q000032:analysis
D014453:Glucuronosyltransferase; D005952:Glucose-6-Phosphatase; D001616:beta-Galactosidase