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,
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
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
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
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
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.
Rong YS, Golic KG. Gene Targeting by Homologous
Recombination in Drosophila. "Science" 2000;288:2013-18.