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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... Full description

1st Person: Wilson, James M.
Additional Persons: Jefferson, Douglas M. verfasserin; Chowdhury, Jayanta Roy verfasserin; Novikoff, Phyllis M. verfasserin; Johnston, David E. verfasserin; Mulligan, Richard C. verfasserin
Source: in Proceedings of the National Academy of Sciences of the United States of America Vol. 85, No. 9 (1988), p. 3014-3018
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Type of Publication: Article
Language: English
Published: 1988
Keywords: research-article
Cell Biology
Gene Transfer
Primary Liver Culture
Hepatocyte-Specific Cytochemistry
Online: Volltext
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Summary: 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.
Physical Description: Online-Ressource
ISSN: 0027-8424

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