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

Gene Therapy volume 10, pages 733–749 (2003)Cite this article

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.

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Acknowledgements

The authors thank Colleen Kelley, Nidhi Mahendru, Tom Miller and Nicole Zandy for their excellent technical assistance. We thank Dr TW Gardner, Penn State University College of Medicine, Hershey, PA, for providing the anti-ZO-1 antibody and Dr FR Boyce, Department of Neurology, Massachusetts General Hospital, Boston, MA, for providing the CMV-lacZ baculovirus and anti-gp64 antibody. We thank Nicole Harhaj for the helpful discussions. This work was supported in part by research grants from the National Institutes of Health (CA23931, CA73045, DK53430, and DK54482 to HCI).

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Authors and Affiliations

  1. Department of Microbiology and Immunology, Milton S. Hershey Medical Center, The Penn State College of Medicine, Hershey, PA, USA

    J P Bilello, E E Cable & H C Isom

  2. Cell and Molecular Biology Graduate Program, Milton S. Hershey Medical Center, The Penn State College of Medicine, Hershey, PA, USA

    J P Bilello & H C Isom

  3. Department of Pathology, Milton S. Hershey Medical Center, The Penn State College of Medicine, Hershey, PA, USA

    H C Isom

  4. Department of Neuroscience and Anatomy, Milton S. Hershey Medical Center, The Penn State College of Medicine, Hershey, PA, USA

    R L Myers

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Bilello, J., Cable, E., Myers, R. et al. Role of paracellular junction complexes in baculovirus-mediated gene transfer to nondividing rat hepatocytes. Gene Ther 10, 733–749 (2003). https://doi.org/10.1038/sj.gt.3301937

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