Gene inactivation by multiphoton-targeted photochemistry

Multiphoton-targeted photochemistry was used to selectively inactivate the expression of genes in vertebrate cells. A membrane permeable DNA-associating vital dye, ethidium bromide monoacetate (visible wavelength single photon absorption peak at 530 nm) was used to photosensitize chromosomes in dividing cells. A 100-ps infrared laser beam operating at 1.06 microns was focused onto a selected region of a mitotic chromosome corresponding to the sites of the nucleolar (ribosomal) genes. Individual cells followed through mitosis demonstrated a reduction in the number of nucleoli formed in daughter cells that corresponded to the number of nucleolar genes sites irradiated. These results demonstrate the ability to selectively manipulate genes by using the focal point specificity characteristic of multiphoton microscopy. This technique should have wide biotechnology applications both in vitro and in vivo.