Zebrafish could become genetics 'lab rat' of choice
WEST LAFAYETTE, Ind. In the post-genomic world, the lowly zebrafish may be king.
Scientists at Purdue University have developed a technique that allows zebrafish to pass genetic modifications to its offspring. The discovery will lead to researchers being able to study genes and proteins in a less expensive way.
The two-inch, black-striped zebrafish known primarily as the last fish living in your kid's aquarium is quickly becoming famous in the scientific world as the best animal to use when studying genetics even better than the mouse.
"Because zebrafish are relatively inexpensive and easy to maintain compared to genetically modified mice, this discovery could greatly accelerate new genetic experiments in vertebrates," says Randy Woodson, director of Purdue's Office of Agricultural Research Programs.
These new experiments would provide information into Alzheimer's, heart disease, certain types of cancer and other diseases.
The zebrafish is an essential tool to a new branch of science called proteomics, also sometimes known as post-genomics. Proteomics refers to the study of an organism's proteins, just as genomics refers to the study of an organism's genetic material. Proteomics is a natural follow-up to the mapping of various organisms' genomes, including the human genome.
"With the human genome project theyre sequencing genes, and each of those genes causes the body to produce various proteins at different times," says Paul Collodi, associate professor of animal sciences at Purdue and primary investigator on the research project. "If you want to understand what the genes actually do, you have to study the function of the proteins they produce, and the zebrafish makes a nice model for that."
Scientists study proteins and gene function by disabling a single gene, and then raising clones of that test subject to see how they develop without the missing gene. Such experiments are called knockout experiments, because the gene is turned off, or knocked out.
Over the past decade, plant scientists have used a small mustard plant, called Arabadopisis, to conduct gene knockout experiments in crops and plants. But until now it has been difficult to conduct knockout experiments in animals.
In mice until now the only animal in which the gene knockout technique works a genetically modified embryo cell, called an embryonic stem cell, is inserted into a developing mouse embryo. An embryonic stem cell is an early embryo cell that has not yet begun to differentiate into various tissues.
The embryo is then transferred into the womb of a female mouse using surgical techniques. After the embryo develops into an adult, some of the modified stem cells will give rise to eggs and sperm that contain the modification. Such a process is laborious and expensive. As a result, transgenic mice can cost thousands of dollars each.
"With mice you have maybe a dozen embryos to work with, and you have to do surgery to transplant the embryos back into the mother," Collodi says. "Compare that to the zebrafish embryo where we can modify 100 embryos an hour, and, because the embryos develop outside the mother, we don't have to do surgery. The entire developmental phase takes only about four days."
Because of the expense and effort required to produce a transgenic mouse, scientists have been searching for another vertebrate animal that would allow these genetic experiments. The technique has been tried in chickens, cows, pigs, sheep and other species of fish without success.
Collodi says the problem has been that, in species other than the mouse, once the embryo cells containing the gene are transferred into a developing embryo and that embryo develops into adulthood, the animal does not produce functional eggs and sperm. Therefore, the gene cannot be passed on to subsequent generations.
"Getting embryonic stem cells to develop into functional eggs or sperm once they were placed in an embryo has been the holy grail for us," Collodi says. "Recently, we've been able to grow these cells in the lab and then transfer them into a zebrafish embryo. We've been able to show that the cells contribute to the germ line of the embryo. That's a big advance for us."
However, Collodi says the next step is to extend the length of time cells are kept growing in the lab so that gene can be inserted. "We've been able to grow the embryo cells in the laboratory for a short time. Now we have to extend the length of time. If we want to make a knockout we have to be able to keep the cells growing in the laboratory for several weeks. Now they last just a few days."
Collodi's technique was published in the Feb. 27 issue of the Proceedings of the National Academy of Science, and was funded by Sea Grant and the U.S. Department of Agriculture.
Mark Hermodson, head of Purdue's Department of Biochemistry, says genetic sequencing information will mean little if the function of the proteins isn't discovered.
"But traditionally thats been fairly unproductive work, to be honest," he says. "The straight genetics approach is to knock out the gene in genetically modified mice and see what symptoms arise. That's very tedious and expensive. Transgenic mice are not cheap."
Zebrafish, on the other hand, can be raised in standard 20-gallon aquariums by the thousands.
Although this is basic research, Collodi says the technique might help advance several fields.
"From an agricultural point of view, transgenics and gene knockouts can be used to control reproduction, disease rate, growth rate and many things that are very valuable in livestock," he says. "This technology could be used to make sterile animals so that transgenic animals cant breed if they escape to the environment."
Sources: Paul Collodi, (765) 494-9280; firstname.lastname@example.org
Randy Woodson, (765) 494-8362; email@example.com
Writer: Steve Tally, (765) 494-9809; firstname.lastname@example.org
Other sources: Mark Hermodson, (765) 494-1637; Hermodson@Aclcb.purdue.edu
Related Web site:
"Production of zebrafish germ line chimeras
Paul Collodi, Chunguang Ma, Lianchun Fan, Purdue University, West Lafayette, Ind.; Rosemarie Ganassin, Niels Bols, University of Waterloo, Waterloo, ON
Although the zebrafish possesses many characteristics that make it a valuable model for genetic studies of vertebrate development, one deficiency of this model system is the absence of methods for cell-mediated gene transfer and targeted gene inactivation. In mice, embryonic stem (ES) cell cultures are routinely used for gene transfer and provide the advantage of in vitro selection for rare events such as homologous recombination and targeted mutation. Transgenic animals possessing a mutated copy of the targeted gene are generated when the selected cells contribute to the germ line of a chimeric embryo. Although zebrafish embryo cell cultures that exhibit characteristics of ES cells have been described, successful contribution of the cells to the germ cell lineage of a host embryo has not been reported. In this study, we demonstrate that short-term zebrafish embryo cell cultures, maintained in the presence of cells from a rainbow trout spleen cell line, RTS34st, are able to produce germ line chimeras when introduced into host embryo. Messenger RNA encoding the primordial germ cell marker, vasa, was present for more than 30 days in embryo cells cocultured with RTS34st cells or their conditioned medium and disappeared by 5 days in the absence of the spleen cells. The RTS34st cells also inhibited melanocyte and neuronal cell differentiation in the embryo cell cultures. These results suggest that the RTS34st splenic stromal cell line will be a valuable tool in the development of a cell-base gene transfer approach to targeted gene inactivation in zebrafish.