Public Release: 

Scientists Find Way To Eliminate Junk Genes In Biotechnology

Purdue University

WEST LAFAYETTE, Ind. -- Two Purdue University scientists have found a way to reduce the guesswork and errors in genetic engineering, and the process has been licensed for commercial application.

The development could speed the delivery of biotech-derived crops, and possibly even benefit human gene therapy, experts say.

Thomas K. Hodges, Purdue's Joseph C. Arthur Distinguished Professor of Botany and Plant Physiology, Leszek Lyznik, a post- doctoral researcher, and colleagues, have developed a two-step process that can insert desired genes on a specific place on a plant chromosome, and then excise other unwanted or "junk" genes that were already on the plant's chromosomes.

Before, when scientists used a variety of methods to insert new genes into a plant's chromosomes, the insertions were random, with genes landing anywhere on the chromosomes, possibly interrupting other gene sequences that coded for vital proteins. In creating crop plants, the result was that often as few as one in a thousand transformed plants might germinate. Even if the plant survived, these random insertions have been a large concern to governmental regulators because they allowed the possibility that the genetic transformation would have unforeseen consequences.

As frustrating and potentially harmful as the problems with random insertions are in plant science, the ramifications of similar problems have virtually stymied animal and human gene transformations. With this new method, the gene can be placed on the recipient chromosome exactly where the scientist desires, eliminating these problems.

For example, a scientist might want to move a gene for drought resistance from sorghum to corn. The new method allows the plant breeder to place the gene exactly on the corn chromosome where it will be most effective, and also gives the breeder the opportunity to remove any corn genes that may be in conflict with the new gene.

The excision part of the new method also will allow scientist to remove other, unwanted genes, such as genes that make cells susceptible to disease.

The Purdue-developed process received a patent in June, and the technology has been licensed to Plant Genetic Systems of Gent, Belgium (a major portion of which was recently acquired by AgrEvo of Frankfurt, Germany). The company has an exclusive license for the technology in Europe and a non-exclusive license in the United States.

Peter Dunn, assistant vice president for research at Purdue and director of Purdue's Biotechnology Institute, says the technology will ease one of the central issues that has slowed the federal government's approval of the first round of genetically modified crops. "If these 'other' genes could be removed from the plant before government regulatory review -- using procedures like those recently developed at Purdue by Tom Hodges and Leszek Lyznik -- it could substantially advance the timetable for approval of transgenic crops," he says.

Ralph W.F. Hardy, president of the National Agricultural Biotechnology Council, agrees that this is a significant development in biotechnology. "The concept of targeted integration of plant genetic material is something that has been anticipated in genetic research for some time," he says. "With a technique that targets specific gene sites, there is less chance that an undesirable change has occurred in the transformed organism."

Although other scientists have developed techniques that also were able to target specific gene sites in genetic transfers, these techniques were limited to the laboratory because they were not practical to apply commercially.

"The fact that this research has received a patent and is licensed indicates that this technique promises to be routine enough to have real application," Hardy says.

John Snyder, assistant director of Purdue's Office of Technology Transfer, says, "We've been able to demonstrate the technical feasibility to the point that companies can see the viability of the technology."

According to Hodges, the technique gives researchers the opportunity to find applications in several new areas. For example, it will allow researchers to remove a defective gene from a plant, change or mutate that gene, and place the improved gene back into the plant on the same site on the chromosome as it was originally found.

As eagerly anticipated and significant as the targeting and gene splicing techniques are, there may be an even more promising application: "The analog of what Dr. Hodges has done in plant genetics is gene therapy in humans," says William Baitinger, director of Purdue's Office of Technology Transfer. "Human gene therapy is behind that of the work in plants, but as you can imagine, the problem of unwanted genes is even more significant in people."

Hodges is enthusiastic about the significance of his finding. "We think of new applications every day," he says. "It's amazing the ramifications this method has."

The first step in the new method is an excision technique that uses a yeast gene identified as "FLP" -- sometimes called "flip" by researchers -- and a segment of DNA that is 48 nucleotides long called "FRT."

The researchers can arrange two copies of the FRT segments on a chromosome so that they bracket the unwanted gene. Then they add the yeast gene to the mix, which produces an enzyme causing the chromosome to break at the sites of the FRT segments.

The chromosome rejoins, but without the unwanted gene, which is digested by the cell.

Because the process uses the yeast FLP gene, scientists sometimes refer to such an excised gene as having been "flipped-off."

The second step allows the researchers to target specific sites on the chromosomes. That new insertion process also has some random gene insertions, but by combining the insertion technique with the FLP/FRT excision technique, the researchers can eliminate all of the random insertions. This leaves a gene that has the desired DNA segment placed exactly where the researchers wanted it, without any random "junk genes."

The process also will allow researchers to remove certain genes that they have inserted, such as herbicide-resistance genes, after the need for them has passed. "This could actually be used in all genetically engineered material released for public consumption," Hodges says.

"This application is also valuable from an environmental or medical perspective. Because it allows us to eliminate antibiotic-resistance genes, for example, it virtually eliminates the possibility of giving rise to the build-up of antibiotic resistance in microorganisms in the environment."

swt/hodges/9611f42

Sources: Thomas Hodges, (317) 494-4657; home, (317) 583-0953; e-mail, hodges@btny.purdue.edu
Leszek Lyznik, (317) 494-8787; home, (317) 463-6107; e-mail, lyznik@btny.purdue.edu
Peter Dunn, (317) 494-6840; home, (317) 463-4363; e- mail, pedunn@ocba.purdue.edu
Ralph W.F. Hardy, home, (705) 887-9887
William Baitinger, (317) 494-2610; home, (317) 743- 1023; e-mail, webaitinger@dsp.purdue.edu
John Snyder, (317) 494-2610; home, (317) 497-1496; e- mail, jrsnyder@dsp.purdue.edu

Writer: Steve Tally, (317) 494-9809; e-mail, tally@ecn.purdue.edu

United States Patent Number 5,527,695
CONTROLLED MODIFICATION OF EUKARYOTIC GENOMES
Inventors: Thomas K. Hodges, Leszek Lyznik, West Lafayette, Ind. Assignee: Purdue Research Foundation, West Lafayette, Ind. ABSTRACT

DNA constructs are provided for the creation of transgenic eukaryotic cells. These DNA constructs allow a more precise and effective transformation procedure by enabling the targeting of DNA sequences for insertion into a particular DNA locus, while enabling the removal of any randomly inserted DNA sequences that occur as a by-product of known transformation process.

Nucleic Acids Research, 1993, Vol. 21, No.4
ABSTRACT
ACTIVITY OF YEAST FLP RECOMBINASE IN MAIZE AND RICE PROTOPLASTS

Leszek A. Lyznik, Jon C. Mitchell, Lynne Hirayama and Thomas K. Hodges, Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907, USA

We have demonstrated that a yeast FLP/FRT site-specific recombination system functions in maize and rice protoplasts. FLP recombinase activity was monitored by reactivation of b- glucuronidase (GUS) expression from vectors containing the gusA gene inactivated by insertion of two FRTs (FLP recombination targets) and a 1.31 kb DNA fragment. The stimulation of GUS activity in protoplasts co-transformed with vectors containing FRT inactivated gusA gene and a chimeric FLP gene depended on both the expression of the FLP recombinase and the presence and structure of the FRT sites. The FLP enzyme could mediate inter- and intramolecular recombination in plant protoplasts. These results provide evidence that a yeast recombination system can function efficiently in plant cells, and that its performance can be manipulated by structural modification of the FRT sites.

Photo captions

Purdue Professor Tom Hodges is the leader of a research team that has developed the first commercially viable technique to eliminate unwanted genes from genetic transfers in crops. (Purdue Agricultural Communications photo by Mike Kerper)

Color photo, electronic transmission, and Web and ftp download available. Photo ID: Hodges/Genes

The new gene excision method uses a yeast gene identified as "FLP" and a segment of DNA that is 48 nucleotides long called "FRT." Researchers place two FRT segments on a chromosome so that they bracket the unwanted gene. Then they add the yeast gene, which produces an enzyme causing the chromosome to break at the FRT sites. The chromosome rejoins, without the unwanted gene. (Agricultural Communications Service graphic by Pam Lassiter and Steve Tally)

Color graphic, electronic transmission, and Web and ftp download available. Photo ID: Hodges/Genes.graphic

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