Needling tissues to accept foreign genes.

Needling tissues to accept foreign genes

Remove the shells from 1,000 housefly eggs by mixing them in a mild bleach. Pour the shelled eggs into a 25-milliliter test tube. Add a healthy dollop of DNA, a few milliliters of water and a pinch of microscopic silicon-carbide needles. Now mix vigorously for 1 minute with a standard laboratory vortex agitator. For variety, try substituting plant tissues or the embryos of other insects.

What sounds like the preparation sequence for a witch's cocktail is actually the recipe for the basic steps in a new gene-engineering technique developed by a molecular biologist at the Agriculture Department's Insects Affecting Man and Animals Research Laboratory (IAMARL) in Gainesville, Fla.

For years, plant and animal breeders have attempted to insert novel genes into unwitting hosts by injecting DNA with a microscopic syringe or by blasting DNA-coated tungsten pellets into biological targets with a special "shotgun" (SN: 5/16/87, p.310). The first technique is arduous, sometimes requiring the injection of 10,000 or more specimens -- perhaps over a year or two--to yield just one that incorporates the new gene and expresses its function. Though shotgunning can potentially target 10,000 specimens at once, this newer technology has proved very costly and rather "disappointing," says Andrew F. Cockburn of IAMARL. "If we shot 10,000 [insect] embryos with the gun, half would be killed outright," he says. And among the survivors, only two or three might incorporate the new material, he adds.

In contrast, Cockburn says, his new "vortex mixing" procedure is inexpensive, using off-the-shelf technology, "and almost ludicrously simple." Though the treatment may kill up to three-quarters of the embryos in any batch, he says, "essentially all that survive will be injected," for a 1,000-fold increase in injection efficiency and a far lower cost. Conceived just a year ago, this was just one of several innovations Cockburn planned to investigate for redesigning the genetic makeup of insect pests. But he recalls that the approach worked so sell the first time it wastried, "I immediately told my technician to drop everything else."

They key to Cockburn's new technique is the use of needle-like, silicon-carbide "whiskers." During mixing, these fibers "punch a lot of little holes" in the target tissues, allowing the infusion of foreign DNA from the cocktail mix, he says.

A few months ago, Gloria Moore, a citrus genticist at the University of Florida in gainesville, began testing the technique on tangerine-like tissues grown in culture. "We've had some encouraging results" suggesting gene incorporation and expression, she says, but it's still too early to be sure.

In the future, Cockburn hopes to improve criteria for such things as optimal mixing times and whisker characteristics. And work by Norbert Perrimon, a geneticist at Harvard Medical School in Boston, suggests such custom tailoring may be essential for certain applications. "Though we had some results which looked kind of promising," Perrimon says, the membranes surrounding fruitfly eggs proved tougher for the whiskers to puncture than expected. The pricking frequency ultimately proved so low that Perrimon has abandoned vortex mixing -- at least for now.