Just months after scientists decoded the complete sequence of the Drosophila (fruit fly) genome, a pair of biologists funded by the National Institute of General Medical Sciences have achieved another significant milestone: the ability to "knock out" fruit fly genes.

"This is something Drosophila scientists have wanted to do for 20 years," said Dr. Kent Golic of the University of Utah, who published the results in the June 16 issue of Science along with postdoctoral fellow Dr. Yikang Rong.

So-called "knock-out" technology is a powerful laboratory method used routinely by scientists working with certain other model systems, such as yeast and mice. But the technology has been sorely missed by the thousands of researchers worldwide who use fruit flies as a research tool to probe mysteries of the biology of plants and animals, including humans.

The new technique offers fly researchers the ability to tease apart the functions of the 13,601 genes in the fruit fly genome. With important genes, Nature has demonstrated an exquisite sense of economy: 177 of the 289 human genes that when "misspelled" are known to cause diseases in people have direct counterparts in the fly.

For decades, fly geneticists have cross-bred strains of flies in order to study their genes. But until now, scientists have not had a way to target a particular fly gene of interest by disabling, or "mutating" that gene.

Thanks to the new work, now these scientists do.

According to Dr. Golic, knocking out fly genes was by no means a sure bet. "It wasn't at all clear that this would work . . . we were scratching our heads about how to find the money to do these experiments," he said.

Dr. Golic's money came from a special NIGMS funding program for "high-risk, high-impact" grants, or R21's as they are called at NIGMS. R21-funded research projects involve high- risk experiments that often have little supporting data but that, if successful, would have a substantial scientific payoff.

Knocking out fruit fly genes fit the bill, according to Dr. Paul Wolfe, a molecular biologist at NIGMS. "This is an outstanding example of a successful R21," he said.

That's one of the reasons the fly research community has been without knock-out technology for so long, Dr. Wolfe added. "No one wanted to take the plunge and take the risk," he said. "Kent Golic did, and it worked."

Knocking Fly Genes Around

Researchers typically employ knock-out technology to create "mutant" organisms that contain a misspelled, and therefore inactive, version of a particular gene that affects behaviors or other features, such as coat color. The method enables researchers to see what happens when the gene is missing.

But Dr. Golic's technique can go either way--his method can also be used to fix faulty genes, or "knock them in," by replacing a defective gene with one that is spelled correctly. This is a key principle underlying gene therapy- -the ability to get rid of the "wrong" copy of a gene and replace it with the "right" one.

That is actually what Drs. Golic and Rong did with fruit flies, working with a fly body color gene called "yellow." The two scientists took an easily recognizable strain of pale-colored flies that are known to have a misspelling in the body color gene that gives normal flies a brownish-black hue. Their goal was to prove that their method could be readily used to exchange genes, by "knocking in" a correct version of the body color gene that would turn the mutant flies brown again.

Drs. Golic and Rong's knock-out/in technique hinges on a common molecular thread that runs through the biological kingdom: Broken bits of DNA don't hang around long--they are swiftly "recombined," or stitched back into the genome.

Dr. Rong capitalized on this chink in the flies' molecular armor, reasoning that if he could purposefully generate an error-free version of the "yellow" body color gene-- containing broken ends--this normal gene would recombine and replace the almost-identical mutant version of the same body color gene sitting within the flies' DNA. This happens easily because only genes with the same (or almost identical) sequence recognize each other to automatically recombine, effectively switching places with each other.

To "knock in" the correct version of the body color gene-- and make the yellow flies revert to their normal brown--Drs. Golic and Rong took a normal, error-free version of the body color gene and modified it slightly for the knock-in procedure.

The only thing different about the normal body color gene that Drs. Golic and Rong used was the presence of molecular "snipping instructions" that enabled the gene to be cut by a specialized pair of enzymes that the researchers also engineered into the flies.

The team then delivered the gene into a test group of flies.

The strategy they used to do so--standard among fly researchers--puts the gene into fly chromosomes at random, a drawback of this current method of introducing genes into flies. This key limitation has hampered fly researchers in the past, because it doesn't permit replacing genes, only adding extra ones--and in no particular location.

Drs. Golic and Rong then introduced into the same flies instructions for producing the necessary molecular sewing implements: two different pairs of genetic "scissors." One of these cuts the inserted gene out of its random position within the flies' DNA, then stitches the gene into a circle. Another pair of scissors is designed to clip this circle of DNA, generating "ripped" ends. To wield control over the scissors, the team engineered the scissor gene so it could be switched on after a short pulse of heat (called a "heat shock").

To replace the wrong body color gene with the right one, Drs. Golic and Rong warmed the flies, switching on the molecular scissors and setting off the cascade of clipping events that culminated in a pair of broken ends of the normal body color gene DNA. The flies' cellular machinery swiftly reacted, directing the broken-ended gene to be sewn into the fly genome and replacing the former, wrong copy of the gene with the laboratory-engineered correct version.

Voila--the next generation of flies, containing the normal version of the body color gene, turned brown.

The same strategy, of course, could be just as easily reversed, so that fly researchers can create mutant flies containing a single wrong copy--designed to replace the right copy--of any gene they wish to study. Such an approach gives scientists the power to determine the functions of genes for which sequence alone is known--and these constitute the vast majority of fly genes.

Reference