Research suggests additional genetic safety measures

By Carol Flaherty

12/01/00 BOZEMAN, Mont. - If you find out one day that your child needs a gene transplant to be cured of a disease, you are going to be very glad that Ed Schmidt has been studying mice.

            For several years, researchers have been using a helpful enzyme to change segments of DNA, potentially including ones that are so flawed they cause disease. Until Schmidt's work, though, no one knew that one of the most useful enzymes used in this work can continue to alter genes after its work is done.

            Schmidt, a researcher in Montana State University's Department of Veterinary Molecular Biology, also has a simple fix for the problem he's found when working with the enzyme nicknamed "Cre." The "fix" is simply to remove it once it has completed its task. After all, says Schmidt, even a plaster cast on a broken arm would cause harm if left on long after the bone was healed. If Cre is not removed, in some cases it could continue to make genetic changes until the chromosomes involved were of no more use than scrambled eggs.

Normal female and male mouse chromosomes at far left, altered female and male chromosomes at right.

        "Researchers thought that you could use Cre to put a gene safely into anything," said Schmidt, "Most people suggested that you could leave this system of making genetic change in place with no ill effects. Our research argues very strongly that Cre should be removed after its work is done."

            Schmidt's research is being published in the Proceedings of the National Academy of Sciences. It was on the World Wide Web Thanksgiving week and will be in the printed issue Dec. 5.

            Dr. Alan Godwin of the University of Kansas Medical Center has reviewed Schmidt's research and says the use of Cre has become very common in genetic research with mice and is being proposed for more and more human genetic studies.

            "The significance of this work is that it shows side effects associated with Cre that aren't predictable, that could lead to cancer or other genetic abnormalities," says Godwin. "It is a cautionary note."

            Cre is an enzyme that comes from "bacteriophage P1," a virus that normally infects bacteria. All of us have a similar enzyme in our bodies that's part of our immune system, says Schmidt.

            Cre has become a powerful tool for manipulating genes in both plants and animals because it naturally prompts a change between DNA elements as a part of its normal viral life cycle.

            This activity that makes Cre so attractive to researchers in the first place is also what creates the risk if Cre is left in the subject.

            The process of using Cre in Schmidt's research is quite simple. Once the target DNA is determined, a researcher or technician injects Cre and a small amount of genetic material into the nucleus of a fertilized egg. Up to 30 percent of the injected eggs will develop into mice with the Cre-introduced genes.

            When Schmidt found the unwanted mutations, he had been trying to develop a mouse that would let researchers better study diseases known to be caused by genetic flaws -- like cystic fibrosis or muscular dystrophy.

            That research failed. However, what Schmidt found is of more immediate importance.

            In the case of Schmidt's work, all male mice descendants of the experimental mice were sterile. Female mice subjects were not affected. The male mice followed the same mating behavior, had normal looking sperm, the same sperm counts and sperm activity. However, all of the eggs these males fertilized died after the first cell division.

Ed Schmidt at a microscope he uses to see mice embryos while introducing new genes into them.

            When Schmidt investigated, he basically found scrambled male mice chromosomes -- at least, that's exactly what it looks like when magnified about 2000 times.

            As it happens, Schmidt's mouse was very sensitive to Cre, but his experiments with it show that future use of this system of making a genetic change need to include the step of removing the system after it has done its work.

            Otherwise, there would be a slow introduction of alterations into the target system.

            "I hope this study will cause people to rethink their approach to genetic therapies," says Schmidt. "I'm not a gene therapy researcher, but my hope is that my research will let these new tools for curing disease be used with greater safety.

Send questions or comments to Schmidt and Carol Flaherty, MSU Communications Services, Bozeman, MT 59717: carolf@montana.edu.

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