Engineered to Glow

two men pose holding small fish tank

Keith Cheng's lab has all the paraphernalia common to cancer labs: glass pipettes and tiny vials, a centrifuge, a fridge with a note forbidding food products. Unlike other labs, however, here an aquarium sits atop one of the clean counters. Inside it swim the office mascots, Bert and Ernie, two lumpy fish the size of fists that look like they were just netted out of the primordial soup. On the door to an adjoining room are taped instructions on what to do in case of flooding—though Cheng's lab is on the seventh floor. The contents of the room make the message clear: dozens of gurgling fish tanks stacked on top of one another from floor to ceiling. Inside them, thousands of fish, 5,000 to be exact, all Danio rerio.

Amateur aquarists everywhere love Danio rerio, better known as zebra fish. Originally found in streams in India, where the waters are always warm and seasonally renewed by monsoons, these tiny vertebrates—the color of quicksilver with dark stripes and big eyes—dart among swaying plants and bubbling castles so quickly it's difficult to keep track of them. Pet-shop owners depend on this hardy species—which often outlasts the delicate angel fish, nervous neon tetras, or disease-prone mollies. But crowd-pleasing zebra fish are gaining an equally appreciative audience among biological researchers. Zebra fish mate easily and often, producing hundreds of eggs bi-monthly; embryogenesis, the development of the embryo to free-swimming fish, takes five days; and zebra fish reach full maturity only three months later. What makes zebra fish most attractive as lab animals, though, is that, unlike white mice or rats, they bear eggs outside of the body. Moreover, the eggs are transparent, allowing researchers to witness each stage of development as the egg becomes a fish. In order to study the developing embryo of a mouse, the maternal mouse would most likely have to be sacrificed. And while Drosophila melangaster, the fruit fly favored by scientists, also lays eggs, the fly is not a vertebrate, which limits its research potential.

Cheng, an associate professor of pathology and an adjunct in biochemistry and molecular biology at Penn State's Hershey Medical Center, began focusing on the genetics of zebra fish seven years ago. Early zebra-fish research, pioneered by the late George Streisinger at the University of Oregon in the 1970s, was a boon for developmental biologists. In a special issue of the scientific journal Development, published in December 1996, photos of a zebra-fish embryo appear in the upper-right hand corner of the magazine; if you flip through the issue, you can see the fish develop from two cells to four, an amorphous sac of cells splitting and forming head, tail, and fins before your very eyes. In the past few years, sophisticated new techniques have encouraged geneticists to study zebra fish as well. Cheng's research group, for instance, is developing zebra fish into an animal model that can detect a certain kind of genetic mutation called a "frameshift mutation," and so help in unraveling the mysterious genetic changes that lead to some human cancers.

tiny fish with lime green lump

In Cheng's lab at midday on a midsummer's day, Erin Gestl, who has both the build and perseverance of a lightweight college wrestler, bends over a tray of vials and squeezes a drop of liquid from a Pipetteman, a biological squirt gun of sorts, into each one. The ultra-whiteness of the walls and the shiny stainless-steel surfaces create a hyper-brightness that seems to hum. Not even the hip-hop music playing in the background can diminish it. Underneath the boom-box and stack of CDs (including Simon and Garfunkel, Bon Jovi, and The Source) hangs an illustration of the plasmid vector Gestl will eventually inject into zebra fish eggs. This illustration—a circle with small sections of it delineated and marked by cryptic abbreviations—looks as if it belonged in the blueprints of a ship. Indeed, it is a blueprint—a map of the very specific genes that will alter embryonic zebra fish in small but significant ways. "Vectors act as a vehicle to move and manipulate DNA," Gestl says, as he mixes different restriction enzymes with a buffer. "They can be inserted into a cell culture or into an animal."

Plasmid vectors are ring-shaped bits of DNA derived from the cytoplasm of bacteria. Restriction enzymes let scientists cut the ring apart at precisely labeled spots. "That's when you can start playing with the DNA," says Gestl, by taking out certain bits and adding others. Afterwards, a different enzyme is used to reform the plasmid into a ring. Genes from any species can be spliced into a plasmid's DNA; then, in theory at least, the engineered ring can be coaxed into the genome of a new species to mutate, or change, it in desired ways. For instance, scientists have used plasmid vectors to insert a gene for herbicide resistance into soybeans. The genetic material that Gestl is working with—the DNA code for a protein that fluoresces either green or blue, depending on the conditions—was isolated from jellyfish. Incorporated into the zebra fish's genome, this ability to fluoresce will make Gestl's transgenic zebra fish powerful tools in detecting cancer-causing changes to a gene.

man looking into microscope

To make a transgenic fish, Gestl injects the vector into the developing fish embryo between the first- and second-cell stages, which causes subsequent cells to include all the genetic information in the vector. Each egg—smaller than a pearl of tapioca—is injected separately with a slender needle finer than a cat's whisker, under a powerful Leica microscope. Gestl says that he's able to inject vectors into 100 eggs per hour. Of those, approximately 30 embryos will accept the new genetic material and fluoresce, 20 will survive to adulthood, but only one or two will have the new genetic information in their germ-line cells (those cells involved in reproduction). These few mutated fish will then be bred with wild-type zebra fish in the hope that the blue-green protein can be transferred to the next generation.

Using the naturally occurring fluorescence found in jellyfish as a marker in live zebra fish is an idea developed several years ago by researchers at the Massachusetts Institute of Technology. Although one might imagine that jellyfish genes would turn zebra fish into underwater fireflies, the fluorescence is far more subtle. It can only be seen under a microscope. "All you have to do is shine a near-UV light. You don't have to hurt the fish," says Gestl. That fact makes it an excellent reporter system: a way to tell if a mutation has occurred. To test the connection between frame-shift mutations and some kinds of cancer, Gestl is also inserting another bit of DNA into the plasmid. Next to the fluorescence gene, he places a repeating unit that is highly sensitive to frame-shift mutations. If such a mutation occurs when a transgenic fish is exposed to a known carcinogen, Gestl and Cheng will know instantly by the fish's glow.

Our goal," says Cheng, "is that all the cells of the fish will fluoresce blue, which means that the fish is transgenic, but the cells will fluoresce green when there are frameshift mutations."

The entire genome, whether of a human or a fish, is coded in the specific language of DNA in each cell. The four DNA bases, ATCG—adenine, thymine, cytosine, and guanine—pair together in a prescribed way. To make a protein, the set of pairs is unzipped and copied onto RNA, which then is translated into the amino-acid sequence of the protein. It's a complex system and, as in all complex systems, mistakes are made. The genetic copier breaks down, skips a beat, repeats a sequence, gives too much or too little. Fortunately, the cell employs proofreading genes that are constantly looking for such mistakes and fixing them. "If you break proofreading genes," says Cheng, "it makes the genome unstable. The genome becomes randomly mutated. Mutations affecting proofreading genes are examples of the mutations I hope to find."

In 1991, Max Planck researcher Christianne Nüsslein-Volhard, a 1995 Nobel laureate, and Harvard researchers Mark Fishman and Wolfgang Driever did a massive screening of zebrafish mutant development by bombarding them with chemicals, radiation, and viruses. "That same year," Cheng explains, "I decided that the zebra fish was the best model to study genomic instability." He came to Penn State in 1992; since then, his lab has produced 12 zebrafish lines carrying instability-causing mutations, with much of the work being done by post-doctoral student Jessica Moore. These fish show elevated rates of additional mutations; one of the problems, Cheng suspects, is an increase in frameshift mutations.

Frameshift mutations, Gestl explains, occur because the cell's genetic copier reads the DNA code three bases at a time, as if each set of three bases were a word. The "word" ATG, for instance, tells the cell to take the amino acid methionine and place it next on the amino-acid string that, when finished, will become a protein. But imagine that before ATG on the code comes AAA—except that the genetic copier made a mistake and only printed AA. If the cell's proofreading genes are defective, perhaps that error won't be caught and corrected. A sequence that should have been AAA-ATG-CTT, for instance, will be read as AAA-TGC-TT: the DNA reader's frame of reference has been shifted, and the message has been garbled. The protein, when made, will likewise be defective, and the fish will be in some way changed. These sorts of mutations, says Cheng, are associated with 10 to 15 percent of human cancers, most notably uterine and some types of colon cancer.

Although hundreds of zebrafish mutants have been produced in other labs—from fish with blood cells that explode when exposed to light to one-eyed fish or fish that can only swim backwards—no one has yet developed a simple test that will determine which genes are responsible for each specific change. Gestl hopes to be the first. He has been perfecting his vector for the last year, a task requiring many trips to the centrifuge, precise timing, and patience. But when his blue-glowing zebra fish suddenly look green, Gestl will be able to say exactly which genes allowed the frameshift error to occur.

Cheng's lab is equipped to handle 8,000 fish. Although there are currently only 5,000 in residence, it's still enough to require constant attention and care. Peggy Hubley, a lab assistant, tends to the fish and nothing else. Hubley formerly worked as a manager of a pet-store wholesaler, overseeing the care of parrots, geckos, lizards, and iguanas. As keeper of the fish, she runs a tight fish lab. Everything must be kept extremely clean—she scrapes algae on a daily basis, even though a central filtering system pretreats all of the tank water. She raises the brine shrimp that are fed to the fish. She's also responsible for treating sick fish. "I talk to them," says Hubley, "especially when they're breeding. I say, C'mon you guys, do your stuff."

The zebra fish live well here, fed and kept with concern. And at night, when all is dark and quiet except for the sound of air gently bubbling through the tanks, perhaps some of them find themselves dreaming, strangely, of jellyfish pulsing fluorescent green in warm waters far away.

Erin Gestl is a Ph.D. student in biochemistry and molecular biology in the College of Medicine, Jake Gittlen Cancer Research Institute, Room C7804, Milton S. Hershey Medical Center, 500 University Dr., Hershey, PA 17033; 717-531-4704; eeg103@psu.edu. His work is partly funded by a grant from the Penn State Geisinger Cancer Center. His adviser, Keith Cheng, M.D., Ph.D., is associate professor of pathology and adjunct professor of biochemistry and molecular biology in the College of Medicine; 531-5635; kcheng@psu.edu. Currently his work is funded by the National Cancer Institute and the National Science Foundation. The American Cancer Society funded earlier zebrafish research.

Last Updated January 01, 1999