The method may be an improvement over conventional identification techniques, which can take months to years. By using a nonviral gene expression system to inhibit the expression of specific genes in zebrafish, the scientists isolated gene function without destroying the gene, says Thomas E. Wagner, distinguished professor of molecular and cellular biology at Ohio University and a principal scientist in the university's Edison Biotechnology Institute. The research was reported in a recent issue of the journal Proceedings of the National Academy of Sciences.
"Our system allows us to stop gene expression long enough to study the gene, then it returns to normal," Wagner says. The technique, dubbed a "knockdown" gene strategy, is much faster than conventional "knockout" methods used to identify gene function. "We're talking a difference between three years with the current methods and two days with ours."
The technique could be valuable to scientists working on the human genome project, a worldwide effort to decipher the sequence of the genetic material present in the human body. Identifying a gene sequence is the first step to identifying gene function, which is necessary for the study of genetic disease.
Although approximately 70 to 80 percent of the human genome has been sequenced, gene identification is moving at a much slower pace, Wagner says. Using conventional methods, genes cannot be identified until their entire sequence is known. The sequence information is used to generate transgenic animals in which the gene of interest is disrupted and made nonfunctional. Researchers then study the differences between these transgenic animals and control animals. The process can take months or years. At that rate, Wagner says, it could take 400,000 work years -- or longer -- to identify the function of the 100,000 to 200,000 genes in the human body.
"Using this knockdown strategy, we only need to know a little of the gene sequence, so that speeds the process a little," Wagner says.
Another cause for the strategy's success is the T7 expression system, which Wagner and other scientists at Ohio University's Edison Biotechnology Center invented several years ago. In order to block gene function, scientists must keep the gene from producing its assigned protein. In the Ohio University studies, scientists used the T7 system to express large amounts of ribozymes targeted at specific genes. Ribozymes are RNA molecules that function as enzymes, binding to mRNA produced from a specific gene and physically cutting the message, and thereby preventing protein production.
"The result suggests that a ribozyme gene knockdown strategy could be as useful as the established gene knockout strategy to identify and elucidate gene function, while being much more efficient," Wagner says.
Until now, studies using ribozymes as the catalyst to stop protein production have worked only in cells. Wagner's research is the first to work in animals. The scientists chose zebrafish instead of mice because of their fast growth rate -- the fish are adult size within 24 hours after they hatch. And there is strong evidence of genetic similarities between zebrafish and other vertebrates, including humans, Wagner says.
"Scientists are learning a lot by the identification of gene sequences, but without information on what those genes do, sequence identification is of little use," Wagner says. "That's why the development of a quick and efficient gene function identification system is so important."
The T7 expression system used in the new gene function identification technique was patented by Ohio University in January 1997, and has been licensed to Progenitor Inc., a biotechnology company in Menlo Park, Calif. Progenitor currently is collaborating with the Edison Biotechnology Institute to use the technology as part of Progenitor's gene discovery program.
A Center of Excellence within the Edison biotechnology Center in Cleveland, Ohio University's Edison Biotechnology Institute is a biomedical/genetics research institute funded in part by the Ohio Department of Development's Thomas Edison Program, a project designed to promote economic development in Ohio.
Co-authors of this new study are Yuefeng Xie, a graduate student in molecular and cellular biology, and Xiaozhuo Chen, an assistant professor in the College of Osteopathic Medicine, both working with Wagner at the Edison Biotechnology Institute at Ohio University.