THE JOURNAL OF GENE MEDICINE RESEARCH ARTICLE J Gene Med 2005; 7: 1097–1112. Published online 6 July 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jgm.764 Functional transfer of eukaryotic expression plasmids to mammalian cells by Listeria monocytogenes: a mechanistic approach Andrea Zelmer 1 Stefan Krusch 1 Andreas Koschinski 2 Manfred Rohde 3 Holger Repp 2 Trinad Chakraborty 4 Siegfried Weiss 1 * 1 Molecular Immunology, GBF, German Research Centre for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany 2 Rudolf-Buchheim-Institute of Pharmacology, Justus-Liebig-University of Giessen, Frankfurter Str. 107, D-35392 Giessen, Germany 3 Microbiology, GBF, German Research Centre for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany 4 Institute for Medical Microbiology, Justus-Liebig-University of Giessen, Frankfurter Str. 107, D-35392 Giessen, Germany *Correspondence to: Siegfried Weiss, Molecular Immunology, GBF, German Research Centre for Biotechnology, Mascheroder Weg 1, 38124 Braunschweig, Germany. E-mail: siw@gbf.de Received: 5 November 2003 Revised: 21 December 2004 Accepted: 4 January 2005 Abstract Background Cystic fibrosis (CF) is one of the most common monogenic disorders in the caucasian population. Gene therapy for CF is principally feasible and bacterial transfer systems might provide novel possibilities for therapy. However, transfection efficiencies are low and need to be improved. Thus, more detailed understanding of the DNA transfer mechanism is necessary to systematically eliminate these restrictions. Methods Functional transfer of GFP-CFTR (cystic fibrosis transmembrane conductance regulator) to eukaryotic cells using attenuated Listeria monocytogenes mediated gene transfer (bacteriofection) was shown by fluorescent microscopy, flow cytometry, immunoblotting and whole cell patch clamping. The characteristics of plasmid transfer were studied by use of electron and fluorescence microscopy, flow cytometry and Southern blotting. Polymerase chain reaction (PCR) was used to screen the genome of bacteriofected cells for cotransfer of chromosomal bacterial DNA. Results Correct intracellular localization and functionality of the GFP- CFTR fusion protein after bacteriofection was shown. Efficient bacterial lysis and release of bacterial content was demonstrated using antibiotics to kill intracellular bacteria. Although only low transfection rates were observed, high numbers of transferred plasmids were detected in host cells under these conditions. However, they were associated with high molecular weight entities and not available to cytosolic transcription. Cotransfer of bacterial genomic DNA was observed in transfectants but occurred at low frequencies. Conclusions In this work we demonstrate that low rates of bacteria- mediated transfection are not due to poor invasion of bacteria, insufficient bacterial lysis, or plasmid DNA degradation. Our data suggest that the transferred plasmid DNA is associated with higher macromolecular structures inhibiting nuclear transport and transgene transcription. Copyright  2005 John Wiley & Sons, Ltd. Keywords CFTR; GFP; L. monocytogenes; gene transfer; bacteria Introduction A large number of diseases are caused by alterations of the genetic informa- tion in germ line. For such monogenically caused dysfunctions, only treatment of symptoms is available at present. Gene therapy, which aims at the somatic Copyright  2005 John Wiley & Sons, Ltd.