New technique for gene transfection using laser irradiation

Background: We have developed a gene transfection system using laser beams. The principle of this procedure is that a small hole is made in a cell mem brane by pulse laser irradiation, and a gene contained in a medium is trans ferred into the cytoplasm through the hole. This hole disappears immediatel y with the application of laser irradiation of the appropriate power. Methods: A pulse-wave Nd:YAG laser with a wavelength of 355 nm was used to make a hole in a cell membrane. To trap a cell, a continuous-wave Nd:YAG la ser with a wavelength of 1015 nm was used, Plasmids that encode the enhance d green fluorescent protein (EGFP) gene were contained in a medium and tran sferred to HuH-7 and NIH/3T3 cells with pulse laser irradiation, We evaluat ed transfection efficiency on the basis of the number of cells that express ed EGFP, Stimulatory protein 2 cells in suspension were fixed using a trapp ing laser and the neomycin-resistance gene was transfected by pulse laser i rradiation. We examined cell proliferation in the selection medium, Results: Cells that expressed EGFP were recognized in the group that was ir radiated by pulse laser. No cells expressed EGFP without irradiation. Trans fection efficiency was approximate to 10% at a plasmid concentration of 10. 0 mug/mL. At concentrations greater than 20 mug/mL, the transfection rate r eached a plateau, We also successfully transfected neomycin-resistance gene s to cells floating in suspension after fixation that was achieved with tra pping laser irradiation. Conclusions: This method enables us to transfect targeted cells, ie, cells in suspension as well as attached cells, with a simple technique that does not involve harmful vectors, The present method is very useful for gene tra nsfection in cellular biotechnology.