Uptake of naked functional DNA into mammalian cells can eukaryotic cells display a temporary increase in membrane be achieved by a number of physical methods. However, permeability. This effect was shown to be caused by cavifor most of these techniques possibilities for therapeutic in tation resulting in the transient generation of cell pores vivo applications -especially to solid organs -are often which allows the direct transfer of naked plasmid DNA. limited. In this report, we describe shock wave permeabilizShockwave transfection of a variety of cell lines was demation as a new physical gene transfer method, which can onstrated. Since shock waves can be well focused within be easily applied, provides great flexibility in the size and particular body regions, future applications of extracorporsequence of the DNA molecules to be delivered, and which ally generated shock waves to tissues simultaneously pershould exhibit an advantageous security profile in vivo.fused with DNA solutions might open up the possibility of Upon exposure to lithotripter-generated shock waves achieving a regionally enhanced in vivo gene transfer.

All tissue culture systems for propagating HBV employed so far make use of tandemly arranged HBV genomes usually under the control of strong foreign promoters. Thus these systems are helpful for virus production but are of limited value in the investigation of the regulation of HBV replication or of the extent to which the expression of viral genes might be influenced by cellular signal transduction pathways. To overcome this barrier we established an HBV-producing cell line (HepG2-4A5) by stably transfecting HepG2 cells with a replication-competent, terminally redundant HBV plasmid (pSPT1.2 xHBV) that contains each of the four major HBV-ORFs only once and exclusively under the control of their own regulatory elements. HepG2-4A5 cells contain a single, nonrearranged, chromosomally integrated, replication-competent HBV genome. In the cytoplasm of HepG2-4A5 cells, all typical viral mRNAs were detectable, but no other viral transcripts were found. Furthermore, all viral gene products are synthesized in a balanced ratio, as close as possible to that found in an in vivo infection. Dane-like particles released from HepG2-4A5 cells were indistinguishable from virions synthesized in vivo, by all physical (electron microscopy, buoyant density) and biochemical (endogenous polymerase reaction, immunogenic behaviour) criteria. Because of the autologous genome organization in this system, the HepG2-4A5 cell line allows studies on the function of the HBV gene products with respect to their involvement in regulating HBV replication under conditions imitating as closely as possible the situation in vivo. Furthermore, this cell line might be a helpful tool in screening antiviral drugs and in studying their effect on regulating HBV replication.

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