Everyone knows that humans are closely related—in terms of our evolutionary development—to monkeys, particularly to Old World genera such as the rhesus monkey. The fact that we share an estimated 93 percent of our DNA with the rhesus macaque has allowed us to learn some important things about ourselves while studying our evolutionary cousin. Although studies of other animals have offered countless insights into the mechanisms of human disease, research into complex disorders such as diabetes, Alzheimer’s disease, and conditions related to aging have begun to call for models with a closer resemblance to ourselves.
Hence the need for ANDi. Named for a reverse rendering of the technique that made him famous (“inserted DNA”), ANDi is the first primate to be born carrying a foreign gene. A healthy young monkey with normal habits of eating, sleeping, and playing, ANDi appears ordinary in every way. A casual observer would never guess that every cell in the animal’s body bears the genetic instructions for producing a glowing green protein.
The protein, designated GFP (“green fluorescent protein”), occurs naturally in jellyfish. Scientists at Oregon Health Sciences University chose GFP as a “reporter” gene for their transgenic project because it would be innocuous to the recipient animal and easily detected if successfully transmitted.
Caption: ANDi (named from “inserted DNA”) is the first primate to be born carrying a foreign gene. (Photo by John Bassir, Oregon Health Sciences University, Portland)
Led by senior scientist Dr. Gerald Schatten at the NCRR-sponsored Oregon Regional Primate Research Center (ORPRC) in Beaverton, the research team started by removing 224 mature egg cells from rhesus females. With the eggs in a culture medium, the scientists introduced the GFP gene into the eggs by means of a viral vector—that is, a debilitated virus that retained its ability to invade cells but was manipulated to transmit the GFP gene instead of a disease. The eggs were then fertilized with rhesus sperm cells, and 40 of the resulting embryos were transferred to 20 surrogate rhesus mothers, two embryos per mother. From this came five pregnancies, including one with twins, but unfortunately, in the end, this arduous, time-consuming process led to just three live births.
Such odds are not unusual in the realm of gene transfer, says Dr. Schatten. “The techniques used for transgenesis in other animals, such as the mouse, probably have an efficiency ranging from 5 percent to 20 percent,” he explains. With monkeys, which usually carry only one embryo at a time rather than a litter of six or eight or more, the number of live births is bound to be much smaller. Despite these limited results, the study achieved its main goal—demonstrating that gene transfer techniques, already familiar in work with animals ranging from frogs to mice to cattle, could now be applied to a primate as well. Not only does ANDi show the presence of the GFP gene in cells sampled from his hair, blood, placenta, and umbilical cord; from the inside of his cheek; and from cells sloughed off in his urine, but cells from the stillborn twins carried the transgene as well—as evidenced by the presence of a green fluorescent protein, as well as by cell analysis.
The twin pregnancy held a special interest for the researchers simply because it is so unusual in this animal. “Twin pregnancies are about 40 times as rare in rhesus monkeys as they are in humans,” says Dr. Schatten. “Those pregnancies are always high risk; only occasionally do they end in live births. Ironically, because the twins in our study were stillborn, we were able to have more access to them for cell sampling and examination than we have with ANDi.”
Caption: DNA encoding a green fluorescent protein (GFP) was packed into harmless virus particles and injected into an egg removed from ANDi’s mother. Inside the egg, the GFP DNA was released and inserted itself into the mother's DNA (small white piece inserted into the large spiral molecule)(Design by Betsy True, University of Wisconsin-Madison)
The procedure at the beginning of the study—injecting all the eggs with the transgene before they had been fertilized—may seem inefficient, but there is sound scientific reasoning behind it. “One reason is to make sure the eggs will be synchronized, so that we’ll know what stage all the eggs are in at any given moment in their development,” says Dr. Schatten. “Another reason is that we think the new gene gets into the mother’s chromosome even before the egg is fertilized. This is important because fertilization can occur quite quickly once the eggs are exposed to sperm; it happens in the first few hours or not at all.” With this approach, once the egg’s chromosomes decondense in the egg’s nucleus during fertilization, a transgene has virtually no chance of inserting itself into the genome.
Continuing his work on the rhesus model, Dr. Schatten has several new goals in mind. He would like to be able to insert a reporter gene that can readily be seen with MRI or PET scanning; if the gene could then be made to target only one type of cell, researchers could follow the development of a single organ or system from its earliest stages. “All this could be done noninvasively, not only on the fetus as it develops, but also on the infant and the mature adult,” Dr. Schatten says. “Imagine if you could image, say, the insulin-producing cells in the pancreas of an animal with diabetes—what insights you might have on the disorder!”
Dr. Schatten is keenly aware of the ethical concerns raised by genetic manipulation of the primate genome. In discussions, he and his colleagues would support national and international regulations of transgenic techniques to limit their use to disease research and treatment. “I try not to minimize ethical concerns, but where these techniques can be used benevolently, we should use them,” says Dr. Schatten. “Further benefit of this pursuit,” he adds, “is that ultimately each rhesus model can provide so much information that fewer animals will be needed.”
Along with his goals he has some hopes, particularly for a major resource that does not yet exist. “Of course, sequencing the rhesus genome would vastly accelerate this work,” says Dr. Schatten.
In the present, though, Dr. Schatten is pleased with the progress of transgenic research, and ANDi himself appears to be a contented creature. “He’s doing fine, living with a couple of buddies, and developing completely normally,” Dr. Schatten says. “He was born in October 2000 and will reach puberty in about four years. In human terms, I would say he’s just past the terrible twos.” Of course, this line of investigation is still young as well. The study that produced ANDi, says Dr. Schatten, “is just a first proof of principle. There’s a lot more work that needs to be done.”
—Sandra J. Ackerman
This research is supported by the Division of Comparative Medicine of the National Center for Research Resources and by the National Institute of Child Health and Human Development.
For more information about the NCRR Division of Comparative Medicine, see http://www.ncrr.nih.gov/comparative_med.asp.
Additional Reading
Chan, A. W. S., Chong, K. Y., Martinovich, C., et al. Transgenic monkeys produced by retroviral gene transfer into mature oocytes. Sciences 291-309-312, 2001.