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Building a better silkworm with piggyBac
By Sharon Guynup

It may be possible to create silkworms with heightened immunity to parasites and viruses, improve the quality of silk and create new super-fibers, or induce the caterpillars to produce silk containing pharmaceuticals. And the same genetic technology may prove valuable in controlling disease-carrying insects and crop pests.

Introducing new genes into silkworms has proved difficult, but now an international research team has found a way to introduce foreign genes into the valuable larvae using a DNA sequence known as piggyBac. Headed by Pierre Couble of the Universite Claude Bernard in France, the team reported the proof-of-concept research in a recent issue of Nature Biotechnology. "The purpose of this experiment was to prove that we had a very effective method of transforming the silkworm," says Couble.

Silk worm moth and cocoon.

Some 4,000 years ago, the Chinese began the multimillenial attempt to modify the silkworm’s genome by domestication and selective breeding. Study coauthor Malcolm Fraser, of the University of Notre Dame, discovered the most recent innovation, piggyBac, a gene sequence he isolated from the genome of a virus called baculovirus. Baculovirus infects insects, and has itself been used to transfer genes.

PiggyBac, a jumping gene, or transposon, has the ability to jump from one place on a chromosome to another. What’s more, any DNA sequence included within the transposon goes along for the ride, making piggyBac a great way to carry genes from one location to another. But transposons don’t just jump willy nilly. Instead, they recognize unique DNA sequences, inserting themselves, and the included gene, very accurately, says Couble.

Having solved the problem of inserting genes into the silkworm’s chromosomes, the team faced another problem. How to get piggyBac and its cargo into cells.

Couble's team found that microinjecting piggyBac into newly-laid eggs worked. They added a marker gene coding for green fluorescent protein to piggyBac, then injected the eggs. The result: glowing green silkworms. Nearly two percent of the larvae growing from injected eggs absorbed the DNA and passed it on as the cells divided. Normally, foreign material, including DNA, is degraded by enzymes in the silkworm egg before the material reaches the nucleus. So although a two percent success rate may sound poor, Couble calls it a success.

The transgenic silkworms have successfully passed the green fluorescent protein DNA to seven subsequent generations. "It's transmitted like a normal trait," says Couble. "The earlier we can insert our foreign gene, the higher the chance of getting it transmitted to the next generation," he adds. He and his team are now trying to duplicate the process, targeting the piggyBac/green fluorescent protein combination to a gene involved in silk production. This experiment could produce a silk that glows green, but that's not the goal.

One long-term objective is to modify the properties of silk—and to create new fibers. For example, the silkworms might be genetically programmed to secrete stronger silk. Conjoining the silkworm's ability to produce an abundance of fiber with the super strength of a protein such as spidroin (the main chemical constituent of spider silk) might produce textiles tough enough for use in bullet-proof vests or parachutes.

Another goal is to engineer a healthier silkworm. The caterpillars are cultivated in many subtropical countries, where high humidity and temperatures encourage a number of harmful viruses. There is great demand from these countries to create a new strain of silkworm that is resistant to the most harmful and prevalent of these pathogens.

Silkworms could also be modified to add proteins of pharmaceutical value to their silk. The protein could then be "harvested" directly from the cocoons. This system would cost very little compared to other methods of animal-based "pharming."

But silkworms may be just the beginning for piggyBac. This advance in the mechanics of introducing foreign genes to silkworms may prove valuable in genetic engineering of many other species. Until now, the methods developed in one insect haven’t worked well in others.

PiggyBac can also transport foreign genes into a number of other insects. It works in the Mediterranean fruit fly, says insect physiologist Paul Shirk and his colleagues at the United States Department of Agriculture’s Center for Medical, Agricultural, and Veterinary Entomology in Gainesville, Fla. They hope to use this technology to render this pest sterile. And a team headed by Martin Klingler, of the Zoologisches Institut, Universität München, reported several month ago in Nature that they have successfully used piggyBac to create transgenic beetles, the first step toward a new way to control these important crop pests. Fraser has used piggyBac in mosquitoes, so it might be applied to the Anopheles mosquito, blocking the insect's ability carry the malaria parasite. The researchers believe piggyBac will work in organisms other than insects as well, including food organisms.

"For 20 years," says Couble, "we were all looking for this miraculous vector that would be universal, that we could use in any species—maybe we’ve found it."

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Toshiki, T. et al. Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector. Nat Biotechnol 18, 81-84 (January 2000).
Berghammer, A.J., Klingler, M. & Wimmer, E.A. A universal marker for transgenic insects. Nature 402, 370 (November 25, 1999).

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