2-10-03
BIOLOGIST OPENS DOORS TO NEW RESEARCH
BY DEVELOPING TRANSGENIC ZEBRAFISH
In his laboratory at Penn State Erie, biologist James Warren continues work that he and colleagues at the University of Michigan began more than seven years ago. Warren and his colleagues have created a line of transgenic zebrafish that will help scientists respond to some of developmental biology's most pertinent questions. In continued research with the transgenic zebrafish, he also collaborates with scientists at the University of Saskatchewan.
"We've developed a strain of genetically altered zebrafish that is now in widespread use throughout the research community," said Warren. "One of our studies has been cited more than thirty times, so we know scientists are building on our findings."
To create the new strain, Warren injected the gene that encoded green fluorescent protein under the control of a heat shock promoter gene into embryonic cells of zebrafish, resulting in a small percentage of the fish incorporating that gene construct into their DNA. Creating the strain required persistence, since only about five in one hundred attempts results in a transgenic fish. After seven years of effort, Warren now has stable strain of zebrafish whose genes, when stressed by heat or other environmental changes, will express green fluorescent protein and glow green under the fluorescent imaging microscope.
Scientists often use zebrafish for studying development because, like humans, they are vertebrates. They are similar to humans in many traits including genes, developmental processes, anatomy, physiology, and behaviors. Female zebrafish produce large quantities of eggs that are easy to maintain, manipulate, and observe, since their eggs are transparent and develop outside the mother's body. In a period of two to four days scientists can watch zebrafish cells divide to form eyes, heart, liver, stomach, skin, and fins.
In two recent papers, Warren and colleagues from the University of Michigan, the University of Saskatchewan, and Tohoku University in Japan used the transgenic zebrafish to demonstrate the real-time effects of pollution and to demonstrate laser-induced gene expression in single cells.
In an October 2002 study published in Environmental Health Perspectives, these researchers used cadmium, one of the most common environmental pollutants, and the transgenic zebrafish to demonstrate visually the effects of pollution at the molecular, cellular, and whole-organism levels.
"It's difficult to study the real-time effects of pollution in humans, because those changes occur over years, and they change physiologic characteristics," he said. "With the strain of zebrafish we've developed, we can expose them to cadmium and observe its effects on the total organism within hours."
The Environmental Health Perspectives study describes work that exposed the embryonic zebrafish to cadmium for 96 hours at a variety of doses and concentrations between their third and seventh days. Observation of the zebrafish found an increased incidence of edema and trunk abnormalities such as spinal kyphosis, scoliosis, and lordosis. At low levels of cadmium, gills fluoresced green. At mid level doses, olfactory and skin cells also fluoresced. As the carefully calculated doses of cadmium increased, the kidney and liver also fluoresced, indicating acute toxicity. "
This is a very visual way to study the effects of pollution," Warren said. "Because the stressed areas of the fish glowed green, we were able to see, under the fluorescent imaging microscope, the real-time effects of a very common pollutant."
In another study, published in 2000 and using the same line of fish, Warren and his colleagues used a laser microbeam to express, or activate, the green fluorescent protein transgene in one cell of the zebrafish. The resulting green fluorescence proved for the first time that scientists could artificially express one specific gene in individual zebrafish cells.
"This technology could be used to decipher the precise roles of individual molecules in zebrafish," said Warren. "We've used this knowledge to investigate the role of individual molecules in normal nervous system development but by extension, this technology could be used to explore potential therapeutic use in cellular regeneration following injury."
Warren's research is supported by the National Institutes of Health and the Paralyzed Veterans of America Spinal Cord Research Foundation, and is done in collaboration with John Y. Kuwada and the developmental biology laboratory at the University of Michigan and Patrick H. Krone and the anatomy and cell biology lab at the University of Saskatchewan.
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Contact:
Loretta Brandon
(814) 898-6063 (O)
(814) 864-9922 (H)
e-mail: lzb6@psu.edu