A method that uses a femtosecond laser to poke a hole in the surface of a single cell and gently tug a piece of DNA through it with optical tweezers offers greater control of gene therapy and genetic engineering.
Developed in the School of Mechatronics at Gwangju Institute of Science and Technology (GIST), researchers used the optical tweezers to grab hold of and transport a plasmid-coated particle to the surface of the cell. Guided by the trapped particle, they created a tiny pore in the cell membrane using an ultrashort laser pulse. While another laser beam detected the exact location of the cell membrane, they pushed the particle through the pore with the tweezers and dropped it into the cell — like a golfer sinking an easy putt.
Optical manipulation of plasmid-coated particles and insertion into the cell through a small pore punctured by a short-pulsed laser. Plasmids produce a green fluorescent protein once inside the cell. (Drawing is not to scale.) Courtesy of Gwangju Institute of Science and Technology.
To determine whether the method had succeeded, the researchers inserted plasmids carrying a gene that codes for a green fluorescent protein. Once inside the cell, the gene became active, and the cell’s machinery began producing the protein. The researchers could then detect the green glow using a fluorescence microscope. They found that approximately one in six of the cells they studied became transfected. This rate is lower than that recorded for some other methods, but those are less precise and involve many cells at a time.
a) A laser scanning microscope image of a cancer cell used in the experiment. The green circles show plasmid-coated particles that have been optically tweezed and inserted into the cell. b) The same cell viewed with a fluorescence microscope. The DNA material inserted into the cell through the transfection process carries a gene that codes for a green fluorescent protein. Here, the cell’s green glow means the transfection process was successful. c) Image (b) superimposed on image (a). Courtesy of Biomedical Optics Express.
“What is magical is that all this happens for one cell,” said associate professor Yong-Gu Lee. “Until today, gene transfection has been performed on a large quantity of agglomerate cells, and the outcome has been observed as a statistical average, and no observations have been made on individual cells.”
Lee hopes the work will allow other researchers to investigate the effects of transfection on individual cells, not just on large populations. With the new technique, “you can put one gene into one cell, another gene into another cell, and none into a third,” he said. “So you can study exactly how it works.”