US scientists
have produced the world’s first genetically altered primate, a rhesus monkey born on
2nd October, 2000, at the Oregon Health Sciences University in Portland. Named ANDi, which
stands for "inserted DNA" spelled backwards, the monkey carries an extra gene
that makes a fluorescent green protein. The achievement was reported in the journal Science.
The scientists believe that the monkey may be a much better model for some human diseases than the most popular model, the mouse. The leader of the team that made ANDi, Professor Gerald Schatten, said that although genetically engineered mice had taught scientists a great deal about human disease, there was a "huge gap" between mice and people.
The mouse has been the favourite and standard model for human diseases because, firstly, it is a mammal and, secondly, scientists have a very fine genetic control of the organism. Mouse genes can be manipulated in a very precise and controlled manner. Individual genes can be totally or partially removed, new genes can be inserted at precise locations, and specific mutations in specific genes, such as those found in human genetic diseases. The modifications can be made such that the effects are only seen in certain tissues. No other organism can be genetically manipulated with this accuracy. The reason for this is that mouse embryonic stem cells (ES cells) have been isolated. ES cells are a special type of cell that found only in the early developing embryo. They are a unique cell type in that they can differentiate (turn into) any cell type in the body. Thus, a whole new mouse can be generated from ES cells. ES cells are cultured in vitro, the desired genetic modification introduced into the cells, then the cells are used to generate new mice such that they carry the genetic modification in all of their cells.
Left: embryonic stem cells
So the mouse has been a very useful model for primary research as well as clinical research. If you don’t know what the function of a gene is, then you can ‘knock it out’ in the mouse and see what happens. If you want to study a human disease, you can introduce the same mutation into the mouse, and often it will display the same symptoms as seen in humans. The mouse can then be used to test potential new drugs and therapies. For some diseases, however, the mouse has not been a good model. The symptoms in the mouse do not match those seen in humans very well. Schatten suggests that transgenic monkeys could fill a crucial hole between mice and people in the study of certain human diseases. "Breast cancer, for example, is poorly modelled in mice because they have no monthly cycle," he says. "Or we could introduce an Alzheimer's disease gene to help test vaccines." Being a primate, they will potentially mimic human conditions far more closely.
So Schatten’s team set out to see if a genetically modified rhesus monkey is possible. The gene they decided to insert into the monkey genome was the gene for the green fluorescent protein (GFP). This is a gene isolated from a species of jellyfish, Aquoria victoria, which codes for the production of a protein which glows green when exposed to ultraviolet light. The gene for the GFP was cloned from the jellyfish, and it has become an indispensable tool in molecular biology. It is used as a cell lineage marker. A cell that carries the GFP gene will glow green under UV light. By inserting the GFP gene into selective cells of an organism, all the progeny cells that have come from the divisions of the original cell(s) will also glow green because they have inherited the GFP gene. The GFP gene can also be fused to another gene of interest such that the protein that is produced from the "fusion gene" glows green wherever it is in the cell, so the location and presence of a molecule in a cell can be tracked over time. The choice of the GFP gene for making a transgenic monkey was purely based on convenience as it was relatively easy to see if the foreign protein was being produced in the transgenic monkey. It was a proof-of-principle experiment. If the procedure is shown to work, then new monkeys may be made that have a human disease gene inserted instead of the GFP gene.
How did they do it and what happened?
The problem faced by Professor Schatten was that no monkey ES cells have been isolated. The only alternative was to use a retrovirus. These are a class of virus that can infect a cell and integrate its own viral DNA into the DNA of the cell. Most people have heard of at least one type of retrovirus - HIV. Molecular biologists use a special type of modified retrovirus called a "pseudo-typed retrovirus". It has been modified such that it retains the ability to integrate into the genome of infected cells but cannot cause disease because its viral disease genes have been removed. By placing a foreign gene (transgene) into a pseudo-typed retrovirus and injecting the virus into the ova of an organism, the foreign gene is integrated into the eggs’ DNA. In vitro fertilization can then be used to produce embryos. Every cell in the resulting embryo carries the foreign gene because every cell comes from the original fertilized ovum. This is the strategy used by the scientists to insert the GFP gene into a rhesus monkey. The problem with this strategy is that there is no control over the site of integration of the transgene. Integration into the genome is random, and this can have unwanted side-effects. If the transgene inserts into another gene, the functioning of that gene can be altered or completely eliminated which may result in developmental defects. The transgene can also integrate into a portion of the genome in which genes are repressed. If this happens, the protein product of the transgene may never be produced.
Left: female rhesus monkey eggs injected with a retrovirus carrying the GFP gene
In practice the procedure can sometimes work but is extremely inefficient. The researchers inserted the GFP gene into 224 unfertilized rhesus monkey eggs. In vitro fertilization produced 40 embryos which were transferred to surrogate mothers. Five pregnancies developed, resulting in three live births. Analysis of DNA from cheek cells, hair, urine and blood samples showed that only one of the three monkeys carried the GFP gene. The young male was named ANDi in recognition of this. Although ANDi’s cells carry the GFP gene, none of the cells in the cheek, hair, urine or blood samples were green under UV light. This implies that although the gene is present, it is not actually producing the glowing green protein it codes for. It could be possible, however, that the GFP gene is expressing the protein in other tissues in ANDi that are not easily accessible in the living monkey. Schatten says other transgenic animals have delayed expressing their transgene product for up to a year after birth. Amongst the failed pregnancies were a pair of miscarried twins. DNA analysis of the twins showed that they were both carrying the GFP gene, but unlike ANDi, their hair follicles and toenails did glow green under UV light. Schatten attributes the miscarriage to the fact that rhesus twins are rare, but the team is investigating whether it might be related to the inserted gene.
Left: the stillborn twins had fluorescent green hair and nails
Although the gene transfer techniques the researchers used are routine in other organisms, reproductive biologist Ted Golos of the Wisconsin Regional Primate Research Center in Madison says the birth of ANDi is the first demonstration that a primate egg can develop normally after such manipulations. "We've made an incremental step from one species to another," Schatten says. And even that small step involved multiple hurdles. Whereas the experiment "is essentially several days' work in transgenic mice," Golos notes, monkey eggs are difficult to collect, and primatologists do not know how to artificially control a monkey’s reproductive cycle. That meant the researchers had to time the experiment precisely so that an embryo was ready when a surrogate mother was at the right stage of her reproductive cycle. In fact, ethics considerations aside, the project might have been easier to achieve in humans, for whom IVF technology is much more advanced.
The announcement of the first transgenic monkey prompted criticism, both from animal welfare groups and those concerned that ANDi represents a step towards genetically modified human beings. "This is yet another step on the slippery slope to designer babies," claimed Dr David King of the Campaign Against Human Genetic Engineering. However, any talk of ‘designer babies’ is premature and ill-informed. This work will not inspire fertility doctors to try the technique with human embryos anytime soon, Schatten predicts. Scientists can’t control where the transgene enters the genome, so the risk of an inserted gene interrupting an important gene would be relatively high. "I don't see an immediate therapeutic application," says bioethicist LeRoy Walters of the Kennedy Institute of Ethics at Georgetown University. Schatten says he does not support any extrapolation of his work to human beings.
Genetic modification of primates does represent one step towards germ-line gene therapy in humans. But only in one sense: technologically. Those who are quick to warn about slippery slopes always concentrate on just one aspect of what is really a complex set of reasons for the development of new medical techniques. Getting over technical hurdles alone is not enough. In medicine, technologies develop not just because there are researchers willing and able to do the work, but because there is an identifiable set of patients who would benefit from a new technique. There needs to be a medical, legal and public impetus in addition to the technology.
The real ethical concern is that this new technology may lead to a dramatic increase in the number of primates used in research. Samantha Gray, a scientific officer with UK group the Fund for the Replacement of Animals in Medical Research, is worried about the trend that the research may set. "After transgenic mice were developed, the use of mice in laboratories rocketed. One of the concerns we have is that once the monkey technique is there, the same thing may happen with primates." She points out that monkeys are unsuitable for much research as they have long gestation periods and produce few young.
But many researchers do not envisage an increase in primate research, both for ethical and financial reasons. "Primate models would be a bit of a mixed bag," says Dave Morgan of the University of South Florida at Tampa, who is using mouse models to develop a human vaccine against Alzheimer’s disease. "Technically they would be very powerful, and clearly they would mimic the human condition far more closely, but I would have concerns over cost and the amount of time it would take to produce results." If researchers are convinced a drug is safe then it would probably be better to jump straight from mice to human clinical trials, he says. And until researchers find more efficient ways to create specific genetic changes, says Schatten, transgenic monkeys will not be common research tools. Even if those techniques were feasible, expense and ethical considerations would limit the use of transgenic monkeys as medical models, he says. "We don't need a knockout monkey for every disease." Schatten believes that a transgenic monkey may be warranted only when there is an existing mouse model for the disease that has already provided a potential therapeutic treatment.
However, some believe that GM monkeys could be useful for understanding illnesses that are difficult to study in rodents, such as those affecting behaviour. Because monkeys are large enough to fit into magnetic resonance imaging machines, researchers might be able to introduce gene markers and track organ development by non-invasive means.
Schatten admits that the jury is still out on how useful primate models will prove. He views such genetic modification as a tool to be used alongside other molecular approaches to fighting disease such as cloning and stem cell research.
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