Retrovirus-mediated transduction of adult hepatocytes

Proc Natl Acad Sci U S A. 1988 May;85(9):3014-8. doi: 10.1073/pnas.85.9.3014.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Cell Line
  • Extracellular Matrix / analysis
  • Genes, Viral
  • Glucose-6-Phosphatase / analysis
  • Glucuronosyltransferase / analysis
  • Histocytochemistry
  • Liver / ultrastructure*
  • Mice
  • Microbodies / enzymology
  • Rats
  • Retroviridae / genetics*
  • Transfection*
  • beta-Galactosidase / analysis

Substances

  • Glucuronosyltransferase
  • Glucose-6-Phosphatase
  • beta-Galactosidase