Single cell/cellular subregion-targeted phototransfection

Introduction of exogenous genetic material, such as DNA, mRNA, siRNA, or miRNA, into cells is routinely performed using one of the many different standardized methods, including lipid-mediated transfection, electroporation, and microinjection to identify their biological function. The ability to control the location and amount of nucleic acids introduced into a cell is particularly important for studying polarized cells such as neurons. Lipid-mediated transfection is simple and fast but lacks regional specificity of delivery, whereas microinjection is regionally specific but labor intensive. To overcome these obstacles, we developed and use the method of phototransfection. In this method, a conventional microscope with a high-power pulse laser of any wavelength is able with minimal destructiveness to induce transient pores into the plasma membrane of the target cell, which remain open long enough to allow exogenous genetic material to diffuse into the cytosol before the pore closes due to membrane dynamics. The technique is not limited by choice of cell type or by genetic material to be introduced, and for RNA allows transfection of multiple mRNAs simultaneously in any desired amount or ratio. Further, phototransfection allows the target area to be a specific cell in a population of cells or a specific subregion within a cell. This protocol summarizes the key steps for performing phototransfection, provides a guide to optimization, and uses as an example green fluorescent protein (GFP) mRNA transfection within a single neuronal process.