Targeted gene transfer in eucaryotic cells by dye-assisted laser optoporation

The blue beam of an Argon laser (488 nm) has been focused on the cell membrane in the presence of phenol-red, an usual component of cell culture media, through a 100 X objective. At the site of the beam impact, due probably to local temperature changes, the cell membrane modifies its permeability. As a consequence of the hit, circular areas, whose radius may be apparently regulated by changing the irradiation time and/or the radiation intensity (energy), appear on the wall, last for a short time and fade spontaneously within 1-2 minutes. No evident signs of cell injury or hurt have been observed afterward. Plasmid DNA, purposely added to culture fluid, easily slips in the cytoplasm; utilizing such approach, thereafter indicated as 'optoporation', we have successfully transfected two genes, namely P-galactosidase and chloramphenicol-acetyl-transferase in murine NIH373 fibroblasts. Therefore optoporation represents an additional procedure for gene transfer with several advantages over already available methods: (1) it only takes advantage of the presence of phenol-red, a normal cell medium component, with no need of addition of extraneous substances; (2) it is a very mild treatment virtually suitable for any cell type and (3) it allows transfection of selected cells even in the presence of cells of different type (providing that they are morphologically distinguishable).