午夜福利1000集合

THE SLIGHT rustling coming from the upside-down polystyrene coffee cups in
the corner of the lab is the only sign that we are down on the farm. Turn over
one of those cups and you鈥檒l see a handful of wiggling creatures, chewing away
on light brown jelly. What looks like an unpleasant case of caterpillar
infestation to you and me, looks like a prototype factory farm to my colleague
William Bentley. Admittedly, it鈥檚 no ordinary farm. These caterpillar livestock
are churning out a valuable medical protein, the anticancer drug interleukin-2

Farming insects for the benefit of humans has an ancient
history鈥攚itness silk production which started as far back as 2500 BC. And
since the 1980s, biotechnologists have been able to manufacture insulin and
other human proteins by inserting the protein genes into bacteria, insect, or
human cells grown in huge steel vats called bioreactors. Now, Bentley, a
chemical engineer at the University of Maryland Biotechnology Institute at
College Park, is combining the best of both approaches in the hope of creating
cheaper, better proteins.

As with all modern farms, the key to success lies in streamlining and
mechanising processes such as sowing (or, more accurately, getting the human
genes into the insect in the first place), husbandry, and harvesting.

When it came to getting human genes into the insects, Bentley turned to an
established technique that takes advantage of one of the scourges of wild moth
and butterfly caterpillars鈥攖he baculovirus. Caterpillars become infected
when they chew on plants that are contaminated with viral particles enclosed in
a protein coat called a polyhedra. In the caterpillar鈥檚 alkaline midgut, the
polyhedra protein dissolves, liberating the viral particles, which then spread
rapidly through the body, turning the insect into a black, gooey, dead mess
within a few days.

Not, however, before the virus has commandeered the insect鈥檚 production
pathways to create more of its own proteins鈥攁 phenomenon that first caught
the eye of biotechnologists in the early 1980s. Then, Gale Smith and Max Summers
of Texas A&M University in College Station realised that they could use the
virus to trick caterpillar cells into making a human protein called
beta-interferon, rather than polyhedra proteins. Beta-interferon is a chemical
messenger of the immune system that has been tested for its potential in
fighting cancer. Smith and Summers simply substituted the beta-interferon gene
for a polyhedra gene in baculovirus, and then used the virus to infect cultured
caterpillar cells, which duly churned out the human protein.

Since then, doctored baculovirus has been fed to a variety of insects,
creating crawling bioreactors like silkworms that make human beta-interferon and
mouse interleukin-3, tobacco budworms that make flu virus proteins, and cabbage
loopers, the insect that Bentley works on, that make adenosine deaminase, the
enzyme that may be missing in some people whose immune systems fail
catastrophically.

Ripe and juicy

But one problem has undermined the use of insects as bioreactors: the timing
of harvesting is both critical, and difficult to judge. The older, bigger
caterpillars make more human protein, but they also contain more protease
enzymes that destroy the protein, and eventually the caterpillar too. What鈥檚
more, the age at which caterpillars produce the most protein depends on
environmental conditions, including humidity, temperature, and how crowded the
caterpillars are in their polystyrene cups. Caterpillar farmers needed some way
of knowing when their caterpillars were at peak ripeness.

Bentley鈥檚 solution is simple and elegant: green fluorescent protein. It makes
jellyfish glow in the dark and has been used since the early 1990s to measure
protein production in a wide range of animals and plants. Bentley joined two
genes together, the one for the interleukin-2, and the gene for the green
fluorescent protein, and attached them to the same control mechanism, or
鈥減romoter鈥�. He inserted this into baculovirus, and fed the baculovirus to his
caterpillars. Then he collected the caterpillars, all glowing green to different
degrees, quickly froze them, and compared the amount of green fluorescent
protein in different samples of mashed caterpillar with the amount of
interleukin-2.

As expected, the more glow, the more interleukin-2 a caterpillar contained,
with the biggest, brightest caterpillars containing roughly 50 micrograms of the
protein, with about one-fifth of that being easy to extract鈥攁 lot of such
a precious protein. Still, just because you can tell when caterpillars are ripe,
it doesn鈥檛 necessarily mean you can rear and harvest them efficiently on the
farm. Caterpillars live in a dog-eat-dog world, so they have to be separated by
size to stop big caterpillars from eating small ones. Just like dogs,
caterpillars need to be fed every day. And at the moment, each ripe caterpillar
must be picked by hand.

So now Bentley, an engineer after all, is working on a way of automating his
caterpillar farm. It may be just a blueprint, but the plans include a honeycomb
of miniaturised feeding pens in which the caterpillars hang from metal tabs, a
detector for monitoring each caterpillar鈥檚 fluorescence, and a means of
electrically shocking individual caterpillars so that they let go of their tabs,
and tumble into liquid nitrogen, killing and snap-freezing them in one fell
swoop.

Priceless

All of which raises the question鈥攊s it worth it? Yes, according to
estimates by the late Marjorie Wier of Biotechnology Transfer in Columbia,
Maryland, a start-up biotechnology company that helped fund Bentley鈥檚 studies.
Interleukin-2 triggers immune cells to divide, and although it鈥檚 still an
experimental drug, it is in demand as a treatment for certain types of cancer.
Interleukin-2 is also an essential component of lab cultures of immune cells
called T-lymphocytes, which are used extensively to study diseases such as
AIDS.

And despite the demand, interleukin-2 is still only available from a few
sources, including extracted from blood or genetically engineered bacteria. Any
product derived from blood carries the risk of disease, and interleukin-2 is at
such low concentrations in blood, it鈥檚 very expensive. Bacteria are no better.
They synthesise interleukin-2 very inefficiently, and 1 milligram of the
substance extracted from genetically engineered bacteria sells for around
$20 000. Compare that with insulin which is very easy to produce in
bacteria; it sells for less than $2 for a milligram. Wier has estimated
that 250 caterpillars could provide as much interleukin-2 as 5 litres of
bacteria, 50 litres of insect cells鈥攐r 1 million litres of human blood.
The cost of factory farming genetically engineered caterpillars is close to
nothing so Bentley鈥檚 interleukin-2 should be far cheaper.

What鈥檚 more, Bentley thinks that caterpillar interleukin-2 could also be
better. Both bacteria and insect cells manufacture their proteins in cellular
structures called ribosomes. Bacteria can use the raw protein straight from the
assembly line, but cells of eukaryotes like humans or insects modify the protein
first by adding complex sugar groups. That means that interleukin-2 from
caterpillars is more like human interleukin-2, and so is likely to be more
active in human cells.

Once Bentley has his cabbage looper caterpillar farm working at full
capacity, he doesn鈥檛 intend to stop with interleukin-2. Working with his
colleague Vikram Vakharia also at the Biotechnology Institute, Bentley plans to
use the farm to manufacture proteins that can be used without removing them from
the caterpillars. Edible vaccines for chicken and fish diseases, are next on the
list. And then, who knows? Chocolate-coated caterpillars for human diseases,
perhaps . . .

* * *

Caterpillars good, maggots better?

WHEN it comes to stocking a human protein farm, fruit fly maggots could prove
even better than moth caterpillars. For starters, fly maggots are happy in
crowded conditions; caterpillars aren鈥檛. In addition, there鈥檚 a wealth of
knowledge about the genetics of the fruit fly Drosophila melanogaster,
which makes it far easier to manipulate its genes.

The fruit fly genome is better mapped than any other complex multicellular
creature, and a tremendous number of its genes have been identified. What鈥檚
more, when human genes are introduced into caterpillars using a baculovirus, the
caterpillars die before they reach sexual maturity. With fruit flies, it鈥檚
possible to create a breeding stock that includes human genes in its make-up.
You inject the human gene connected to a gene that makes eyes orange, into
white-eyed fly embryos. When the flies reach adulthood, you breed them with
other white-eyed flies. Any orange-eyed offspring will contain the human gene in
all their cells, including egg and sperm cells, and they can be used to create a
colony that makes human protein.

With these advantages in mind, my colleague William Bentley and I, are hard
at work at the University of Maryland Biotechnology Institute in College Park
making fruit flies that can synthesise human medicinal proteins like
interleukin-2 even more efficiently than caterpillars. We have created our
鈥渃onstruct鈥濃�攖he genetic sequence that will be injected into the fruit fly
embryos. It includes the orange-eyed gene, the gene for interleukin-2, and the
gene for the protein that gives jellyfish their eerie glow. The fruit flies
containing the most interleukin-2 will glow brightest green.

Now, we are linking this construct to different promoters, the sequences that
regulate a gene鈥檚 activity. The plan is to use the promoters to create flies
that manufacture the protein in different tissues, at different ages, and under
different environmental conditions, and see which produce the interleukin-2 most
efficiently. For example, fruit flies have a lot of gut tissue and body fat, so
we鈥檒l create flies that make interleukin-2 in either of these tissues, and see
which generates the most interleukin-2.

Once we have optimised our engineered fruit fly, we hope to go one better: we
will irradiate flies to create mutants, breed the mutants with our engineered
flies, and then test their offspring鈥檚 ability to produce interleukin-2. The
stars will be used to breed flies with super-enhanced levels of
interleukin-2.

For all you ever needed to know about fruit fly
genetics see http://flybase.bio.indiana.edu