Research Article

Gene Therapy (2003) 10, 733–749. doi:10.1038/sj.gt.3301937

Role of paracellular junction complexes in baculovirus-mediated gene transfer to nondividing rat hepatocytes

J P Bilello1,2, E E Cable1, R L Myers4 and H C Isom1,2,3

  1. 1Department of Microbiology and Immunology, Milton S. Hershey Medical Center, The Penn State College of Medicine, Hershey, PA, USA
  2. 2Cell and Molecular Biology Graduate Program, Milton S. Hershey Medical Center, The Penn State College of Medicine, Hershey, PA, USA
  3. 3Department of Pathology, Milton S. Hershey Medical Center, The Penn State College of Medicine, Hershey, PA, USA
  4. 4Department of Neuroscience and Anatomy, Milton S. Hershey Medical Center, The Penn State College of Medicine, Hershey, PA, USA

Correspondence: DR HC Isom, Department of Microbiology and Immunology H107, Penn State College of Medicine, Penn State Milton S. Hershey Medical Center, PO Box 850, 500 University Drive, Hershey, PA 17033-0850, USA

Received 28 April 2002; Accepted 15 October 2002.

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Abstract

Gene delivery to differentiated hepatocytes is notoriously difficult. Hepatocytes plated on collagen-coated dishes and maintained in dimethyl sulfoxide (DMSO)-supplemented medium acquire paracellular junctions, arrange themselves in multicellular islands and are an excellent in vitro model for studying liver function. Baculovirus-mediated gene delivery to hepatocytes in this culture system is restricted to peripheral cells of the islands. However, this limitation can be overcome by transient calcium depletion of the cells prior to and during baculovirus infection. Examination of the mechanism underlying this process revealed that calcium depletion was accompanied by a transient loss of intercellular contacts and paracellular junction complex integrity, increased distance between adjoining cells, and internalization of the tight junction protein, zona occludens ZO-1. Internalization of ZO-1 was accompanied by baculovirus infection of internal cells of hepatocyte islands. When calcium levels were restored, paracellular junction complex integrity returned to normal by 12 h. No permanent alterations in hepatocyte ultrastructure and albumin mRNA, and protein expression were caused by this gene transfer method. Loss in paracellular junction complex integrity exposes the basolateral (sinusoidal) surface of hepatocytes resulting in homogeneous baculovirus-mediated gene delivery to approximately 75% of the cells in long-term DMSO culture. We conclude that the use of recombinant baculovirus as a vector in combination with transient calcium depletion is a highly efficient method for delivering exogenous genes to hepatocytes without loss of hepatic differentiation.

Keywords:

baculovirus, hepatocyte, paracellular junction complexes, gene transfer, calcium depletion