Although geneticists use a variety of gene transfer methods to introduce foreign DNA into microbial, plant, and animal cells, many important organisms do not respond to these established techniques. Problems with delivering genes in a reproducible, cost-effective, and timely manner still preyent researchers from manipulating certain genomes.
But a new transformation technique, called particle gun technology, has overcome many of the obstacles of existing techniques and holds promise for becoming a universal gene delivery system and a means to introduce drugs and other biologically itnportant substances into cells.
First developed in 1984 by John Sanford, Edward Wolf, and Nelson Allen at Cornell University in Ithaca, N.Y. particle gun devices propel millions of DNA-coated particles past rigid plant cell walls and delicate membranes, allowing direct deposit of genetic material into living cells, intact tissues, and microscopic organelles.
Gene guns operate on the principle that under certain conditions, DNA and other genetic material become “sticky,” readily adhering to biologically inert particles. By accelerating this DNA-particle complex in a partial vacuum by any number of possible mechanical systems and placing the target tissue within the acceleration path, DNA is effectively introduced. While it’s not known exactly how many researchers are developing their own gene gun designs, Du Pont Co., Wilmington, Del., and Agracetus Inc., Middleton, Wis., have United States patent applications pending.
The Biolistic gene gun, developed by the Cornell group but recently acquired by Du Pont (see table), accelerates particles in a manner similar to the way a standard gun propels a bullet. The device uses a small gunpowder charge and a macroscopic projectile about the size of a .22-caliber bullet to accelerate DNA-laden tungsten particles. The particles are located on the front, flat surface of the plastic cartridge. When the gunpowder explodes, the “bullet” carrying the particles travels down a smooth, six-inch barrel and hits a plastic stopping plate. On impact, the bullet fuses with the plate, but the small tungsten particles pass through to the target tissue via a hole in the center of the plate.
The Particle Gun, developed by Dennis McCabe at Agracetus, uses a high-voltage discharger to vaporize a water droplet. The resulting shock wave from the explosive vaporization causes a thin metal sheet containing DNA-laden gold particles to rise and accelerate toward a mesh screen. The screen retains the metal sheet but passes the particles to the target tissue. According to Paul Christou, senior scientist at Agracetus, the intensity of the electric discharge can be adjusted to control the penetration of particles into the target tissue.
Although still in its infancy, particle gun technology represents a major leap forward in genetic engineering. “No existing technique offers such a rapid and practical method of inserting genetic material into such a wide range of cells and tissues,” says .Sanford. “The protocol is simple and is essentially the same, regardless of DNA or target cell.”
The technique is especially exciting for researchers working in the field of plant genetics from both a commercial and a pure research perspective, says Anthony Cashmore, director of the Plant Science Institute at the University of Pennsylvania in Philadelphia.
“It used to be difficult, if not impossible, to transform corn, wheat, barley, and other cereals with Agrobacterium tumefaciens,” a parasitic organism found in soil that is commonly used to infect tobacco, tomato, and other dicot plants with foreign DNA, says Cashmore. “But with the gene gun, transformation of the world’s major cereal crops into plants that are resistant to pathogens and herbicides is now a distinct possibility.”
The direct introduction of genes into intact plant cells surpasses the electroporation method of transferring genes because it eliminates the need to regenerate whole plants from a protoplast (a plant cell with its cell wall removed). With electroporation, protoplasts immersed in a solu tion of foreign DNA are subjected to an electric field. The current destabilizes the membrane, allowing DNA to enter the cell. While it is easy to remove plant cell walls for this process, it is very difficult to regenerate the transformed protoplast into a plant. In contrast, the gene gun uses intact plant cells, which scientists have been reasonably successful in regenerating.
Another important use of the DNA gun involves the transformation of organelles. For the first time, researchers have transformed yeast mitochondria and the chioroplasts of Chiamydomonas (algae) using this technology. The ability to transform organelles is significant because it enables researchers to engineer organelle-encoded herbicide resistances in crop plants and to study photosynthetic processes.
The focus of Cashmore’s research is on how light regulates the expression of genes in photosynthesis. He now uses Agrobacterium to transform key genes in tobacco plants to note the effect changes in gene structure have on gene expression. Each of Cashmore’s experiments, which involve isolating the genes, introducing them into tobacco cells, and regenerating the cells to plants, takes from four to six months. But by redesigning his experiments using the gene gun to in- troduce plasmids into cells, he estimates that he could assay for transient gene expression within a few days of transformation.
While many important potential applications of particle gun technology have yet to be explored, a technology exchange program located at Cornell’s Plant Science Center and funded by the National Science Foundation is making it easier for researchers to investigate this new technique. The facility demonstrates the Biolistic process and offers assistance in designing and testing experiments to researchers by appointment. There is no charge for this service (see table).
Carole Gun is a freelance science writer based in Philadelphia.