|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. (Image courtesy of the Gwangju Institute of Science and Technology)|
Current methods for inserting DNA into living cells don’t allow precise control of how and when to insert it or require burning through large numbers of cells before getting it into one cell. Recognizing this, a team of scientists at the Gwangju Institute of Science and Technology (Gwangju, South Korea) has developed a method that allows them to precisely poke holes on the surface of a single cell with a high-powered femtosecond laser and then gently tug a piece of DNA through it using optical tweezers, which draw on the electromagnetic field of another laser.
“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,” says Yong-Gu Lee, an associate professor in the School of Mechatronics at the Gwangju Institute of Science and Technology in South Korea and one of the researchers who carried out the study.
|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). (Image courtesy of Biomedical Optics Express)|
In the new study, the researchers sought to safely transfect an individual cell. To manipulate the foreign DNA, the scientists used optical tweezers, which essentially tweak a laser beam whose electromagnetic field can grab hold of and transport a plasmid-coated particle. The researchers first moved the particle to the surface of the cell membrane. Guided by the trapped particle, they then created a tiny pore in the cell membrane using an ultrashort laser pulse from a femtosecond laser. While another laser beam detected the exact location of the cell membrane, they pushed the particle through the pore with the tweezers. Using this technique, the scientists were able to ease a microparticle right up to the pore in the membrane and drop it into the cell.
To determine whether their 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.
Lee hopes the work will allow other researchers to investigate the effects of transfection on individual cells, not just 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 says. “So you can study exactly how it works.”
The team’s approach has been published in the journal Biomedical Optics Express; for more information, please visithttp://www.opticsinfobase.org/boe/abstract.cfm?uri=boe-4-9-1533.
Subscribe now to BioOptics World magazine; it’s free!