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 β-galactosidase. Retrovirus-mediated transduction was measured by two methods: (i) an in situ cytochemical stain that specifically detects the expression of viral expressed β-galactosidase, and (ii) Southern blot analysis, which measures the relative copy number of integrated provirus. Maximal transduction efficiency of ≃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 >95% of cells were hepatocytes. The demonstration of hepatocyte-specific organelles in cells expressing the viral-directed β-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.
|Original language||English (US)|
|Number of pages||5|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|State||Published - 1988|
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