DNA-loaded bacterial ghosts efficiently mediate reporter gene transfer and expression in macrophages.
There is a demand for efficient and safe DNA delivery vehicles mediating gene transfer and expression. We present bacterial ghosts as a novel platform technology for DNA delivery and targeting of macrophages. Bacterial ghosts are cell envelopes of gram-negative bacteria that are devoid of the cytoplasmic content. Escherichia coli ghosts were loaded with plasmid DNA and linear double-stranded DNA. Confocal laser scanning microscopy and flow cytometry confirmed that the DNA localized to the inner lumen of bacterial ghosts and was not associated with the outer surface of the bacteria. Up to approximately 6000 plasmids could be loaded per single ghost and the amount of loaded DNA correlated with the DNA concentration used for loading. E. coli ghosts loaded with plasmids encoding the enhanced green fluorescent protein (EGFP) targeted efficiently murine macrophages (RAW264.7) and mediated effective gene transfer. The EGFP was expressed by more than 60% of the macrophages as measured by flow cytometry detecting the green fluorescence and immunocytochemical staining with antibodies specific for EGFP.
Paukner
Susanne
S
Institute of Microbiology and Genetics, Vienna University Biocenter, Dr. Bohrgasse 9, A-1030 Vienna, Austria. susanne.paukner@gx.novartis.com
Kudela
Pavol
P
Kohl
Gudrun
G
Schlapp
Tobias
T
Friedrichs
Sonja
S
Lubitz
Werner
W
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology*; D004247:DNA / Q000008:administration & dosage* / Q000032:analysis / Q000235:genetics*; D004926:Escherichia coli / Q000166:cytology*; D005434:Flow Cytometry; D015870:Gene Expression / Q000235:genetics; D018014:Gene Transfer Techniques; D017930:Genes, Reporter / Q000235:genetics*; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000008:administration & dosage / Q000032:analysis / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D004247:DNA