TARGETED GENE-TRANSFER IN EUKARYOTIC 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 cul ture 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 an d/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, pur posely added to culture fluid, easily slips in the cytoplasm; utilizin g such approach, thereafter indicated as 'optoporation', we have succe ssfully transfected two genes, namely P-galactosidase and chlorampheni col-acetyl-transferase in murine NIH373 fibroblasts. Therefore optopor ation represents an additional procedure for gene transfer with severa l advantages over already available methods: (1) it only takes advanta ge 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 t reatment virtually suitable for any cell type and (3) it allows transf ection of selected cells even in the presence of cells of different ty pe (providing that they are morphologically distinguishable).