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Mice GM with jellyfish genes glow in the dark NS 22 May 1999


Look, no new genes A subtle technique tricks plants into doing their own genetic engineering
NS 31 July 99

SUPPOSE you could modify the DNA of plants without having to introduce a package of genes from other organisms. Charles Arntzen of Cornell University in Ithaca, New York, says that you can-and that his team has already demonstrated the technique by creating tobacco plants resistant to a widely used class of weedkillers. Given that the furore surrounding genetic engineering stems in part from concerns about effects of "foreign" genes, Arntzen hopes the new technique, dubbed chimeraplasty, will help quell public fears. However, [email protected] opposed to genetic engineering remain sceptical. in conventional plant genetic engineering, the gene for the desired trait can come from a completely unrelated specieswhich doesn't even have to be another plant. The gene is introduced with a package of genetic "switches" to ensure that it continues to work in its new host. Opponents of genetic modification argue that exchanging genes between species which cannot naturally interbreed could have unpredictable and potentially damaging consequences in the environment. More controversially, they have seized upon experiments conducted by Arpad Pusztai, formerly of the Rowett Research Institute in Aberdeen, which suggested that the genetic switches introduced with a foreign gene can harm animals that eat engineered plants (This Week, 20 February, p 4). While most experts have dismissed these conclusions, pointing to flaws in Pusztai's experiments, the alarm they generated still lingers. Rather than introducing foreign genes, the new method subtly alters a plant's own genetic sequence. The key tool, developed by the company Kimeragen of Newtown, Pennsylvania, is a hairpin-shaped loop of 25 bases, or letters of the genetic code, chosen so that it binds tightly to the two strands the target gene, except at one point where the base lining up with each strand of the gene's DNA doesn't match. The hairpins are genetic "chimeras", because they contain both RNA and DNA. "The RNA is included because it stabilises the hairpin," says Arntzen. It also helps the hairpin bind more tightly to the gene. Arntzen coats microscopic gold particles with the hairpins which are then fired into plant cells. "In the nucleus, they fit themselves between two strands of DNA," he says. The bases that don't match with the target gene's sequence then fool the plant's own DNA repair enzymes into altering the target gene (see Diagram). They replace the apparently mismatched bases so that they bind correctly to the hairpin. Over time, the hairpin decays away, leaving the gene with an altered sequence. Arntzen has used this process in tobacco plants to change the gene for a vital enzyme called acetolactate synthase, which is disrupted by sulphonylurea weedkillersAfter they changed the gene so that it made a version of the enzyme with one altered amino acid, the plants were able to resist the weedkillers (Proceedings of the National Academy of Sciences, vol 96, p 8774). Because it can only make subtle changes to a plant's DNA, Arntzen admits that the technique is less versatile than conventional methods of genetic engineering. But he predicts that it could be used to tweak crops so that they are more nutritious, producing extra vitamin E, for example. Even though no foreign genes are involved, groups opposed to genetic engianeering are still wary. "We don't know enough about whether the altered gene affects other genes, and what the effects of that would be," says Adrian Bebb of Friends of the Earth. Andy Coghlan

Vive la difference
NS 17 Apr 99
A unique alliance is racing to map genetic variability

THE era of personalised medicine, when patients will be prescribed drugs tailored to their precise genetic make-up, has come a step closer. This week, 10 major drugs companies and the Wellcome Trust, the world's largest medical charity, are unveiling a plan to map the variability of the human genome within two years. The data will immediately be made public to head off attempts by other companies to patent the information for their own gain. The $45-million project will identify and analyse single nucleotide polymorphisms (SNPs). These are variations in single DNA bases, which account for most of the genetic differences between people. SNPs are thought to be a major determinant of people's susceptibility to disease and their response to drugs. The ongoing Human Genome Project, which aims to have a working draft of our entire genetic blueprint by February next year, will produce a "consensus" sequence for the typical human. But David Bentley, head of genetics at the Wellcome Trust's Sanger Centre near Cambridge, argues that individual variation is even more important. Up to a tenth of the three million SNPs thought to exist will be mapped by the new consortium. "It will be a representative map," says Arthur Holden, former head of British biotechnology company Celsis. Holden will be chief executive and chairman of the consortium. The Wellcome Trust is providing $14 million for the project. The rest will be made up from equal contributions by the 10 companies: AstraZeneca, Bayer, Bristol-Myers Squibb, Hoffman-La Roche, Glaxo Wellcome, Hoechst Marion Roussel, Novartis, Pfizer, Searle and SmithKIine Beecham-Holden hopes to convince other companies to join the consortium. An original proposal for an SNP map from the British pharmaceuticals giant Glaxo Wellcome evolved into a consortium when the companies agreed that they would make more rapid progress by first collaborating on identifying SNPs and then competing on efforts to make drugs using the information. "To my knowledge something like this has never been done before," says Allen Roses, head of genetics at Glaxo Wellcome. The Sanger Centre, the Washington University School of Medicine in St Louis and the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts, will be responsible for identifying SNPS. Those that look as if they might be medically important will then be mapped by the Human Genome Center at Stanford University in California and the Sanger Centre. Finally, the information will be analysed and built into an overall genome map by the Cold Spring Harbor Laboratory on Long Island, New York. By making the information freely available, the consortium hopes to prevent other companies from patenting key SNPs. But it could find itself in a race against firms that have decided to go it alone. Genset, based in Paris and San Diego, says it intends to build its own SNP database and file patents. Celera Genomics of Rockville, Maryland, is also investing heavily in SNPS. However, the consortium has already made a start. The Sanger Centre has completed a three-month pilot project which mapped SNPs on chromosome 22. "There are lots of SNPs already in the bank," says Bentley. Maft Walker

Supercell NS 24 Apr 99

Endless supplies of living tissue, grown from a single cell and ready to repair damage anywhere in the body-Philip Cohen asks how close the dream is to reality

A RARE, deadly cancer seems a strange place to begin thinking about a range of revolutionary life-giving treatments, but then everything about teratocarcinomas is, frankly, bizarre. These tumours grow from the testicles and ovaries and can sprout gut and muscle tissue, nerves, teeth, tuffs of hair-even grotesque facsimiles of fingers and legs. In different cultures and times, teratocarcinomas have been taken as a sign that a person dabbled in the occult, indulged in sexual sins or even as an omen of future prosperity. In reality, teratocarcinomas grow when the mother of all cells, an embryonic stem cell (ESC), runs amok. ESCs appear a few days after an egg is fertilised, and aren't supposed to hang around for long. At this early stage the egg, called a blastocyst, is just a hollow ball floating down the oviduct to the uterus. The cells on the outside of this freewheeling ball will become the placenta, but it is a small mass of nondescript cells towards the inside that will grow into the embryo proper. These are the ESCS, which quickly begin to grow and divide, becoming progressively more specialised until their fates are sealed as a particular tvt)e of cell-a heart cell with its own rhythmic beat, a neuron with its long thin axons, or a phagocytic immune cell. Ultimately, every cell in the body can be traced back to an ESC (see Diagram, p 34). Unless, of course, something goes wrong. Then, a few ESCs stay in their undifferentiated state until some unknown trigger makes them spin out of control spawning wrong types of tissue in the wrong place-a teratocarcinoma. Until recently, teratocarcinoma cells were the closest biologists had come to capturing human cells that are pluripotent, endowed with the awesome potential to create any one of the human body's 200 or so tissues. That, however, has all changed in the past few months.

Rapid results

In quick succession, researchers have shown that it is possible to pluck ESCs from human embryos and fetuses and grow them indefinitely in a flask. As if that wasn't enough, the dogma which says that once a cell has taken on its adult task it cannot then switch careers, is falling apart. First, there was Dolly, a whole sheep created from a mature mammary gland cell. And now it seems that with much less technical intervention, mouse nerve cells will transmogrify into muscle cells, and human marrow cells into nerve cells. Hamess the ability of cells to make new tissue types, or switch tissue types, and the impact would be huge. People die every day for want of donor tissue. In theory, a single tube of cells could be turned into enough tissues to treat hundreds or thousands of patients, filling freezers with ready-to-use immune cells, neurons, heart or liver tissue. Having these healthy body parts at the ready could revolutionise the treatment of Alzheimer's and Parkinson's disease, stroke and spinal cord injuries, heart disease and diabetes. As Harold Varmus, director of the National Institutes of Health, told the US Congress, "There is almost no realm of medicine that might not be touched by this innovation." Still, although scientists are willing to talk a reporter's ears off about the potential of these discoveries, ask them how they intend to harness this power, and they go strangely silent. Hardly surprising given that the bragging rights and lucrative patents will fall to whoever stakes the surest claim on the answer. What they are willing to tell you is certainly not a trade secret: it won't be easy.

But nor was tracking down the ESCs in the first place. Most people assumed that ESCs lasted little more than eight days, so their only possible source was thought to be embryos discarded by IVF clinics. But they are hard to come by, and in the US, the government forbids spending federal research dollars on human embryos. Despite the difficulties, last November, after six years of trying, a team led by James Thomson at the University of

Wisconsin, Madison, reported that it had captured human ESCs from emrbryos. In addition, researchers headed by John Gearhart at Johns Hopkins University in Baltimore made the startling announcement that they had captured human ESCs from nine-week-old aborted fetuses. Gearhart's approach was a long shot, based on a finding, by Brigid Hogan of Vanderbilt University in Nashville, Tennessee, that in mice some ESCs survive in the gonads beyond the earliest stages of development. As expected, the human ESCs had many of the same surface proteins as human teratocarcinoma cells, confirming their common origin. And when the researchers injected the ESCs into mice, they formed teratocarcinomas that sprouted cells from each of the body's "germ layers" (Science, vol 282, p 1145; Proceedings of the National Academy of Sciences, vol 95, p 282). These three classes of tissue-the ectoderm, endoderm and meso derm-emerge at the first step of cell specialisation and give rise to the ner vous system, internal organs and musculoskeletal system (including the blood) respectively. The second discovery, that brain tissue can transmute into blood, also began with a tumour, one called a medulloblastoma which attacks the brains of children. Occasionally these tumours contain muscle cells, which were thought to migrate into the brain from outside. "Then we had this bizarre idea," says Angelo Vescovi of the National Neurological Institute in Milan, Italy. "What if these cells could somehow leam to switch their identity?" Vescovi's team tested their idea on neural stem cells, the least spe cialised cells in the brain. When they injected these cells into the bone marrow of mice that had most of their own white blood cells des troyed, they turned into new white cells (Science, vol 283, p 534). This was the first clear demonstration that, with minimal intervention, nor mal cells from an adult animal can spawn cells from more than one germ layer. "It's simply remarkable," says Irving Weissman, a cell biologist at Stanford University in California. "I'm sure other labs are rushing to do similar experiments right now." There are already hints that cells in the bone marrow of humans are capable of equally dramatic changes. Last year, Darwin Prockop of MCP Hahnemann University in Philadelphia showed that when human stromal marrow cells, which give rise to muscle and connective tissues, are injected into the brains of rats, they behave just like nerve cells, migrating deep into the brain (Proceedings of the National Academy of Sciences, vol 95, p 3908). Later this year, Prockop's team plans to publish conclusive genetic evidence that the marrow cells have turned into nerve cells. "We have some truly dramatic results," he promises. Just because cells from our bodies can perform these remarkable feats doesn't mean we can easily mimic the processes in the lab. While it's true that ESCs have the potential to become every cell type, they need persuading. The embryo, for example, has an elaborate set of whips and carrots that compel cells to grow, alter their fate or die. These include "growth factors"-molecules that come first from the outer layer of the blastocyst, and later from other cells within the embryo. Then there are physical forces such as being compressed or twisted, and even electrical charges from neighbouring cells.


Under these influences, some genes turn on in a developing cell and others off. This changes the cell's characteristics, including the next set of whips and carrots it will have to respond to. Similar cues must persuade brain cells that are moved to the bone marrow to turn into blood cells. Given the daunting complexity of the signalling system, you might expect the researchers to be a gloomy bunch. Nothing could be further from the truth, judging by the money pouring into biotech companies working on stem cells. Besides California-based Geron, which backed Gearhart and Thomson's work, Advanced Cell Technology in Worcester, Massachusetts, and Stem Cell Sciences in Melboume, Australia, say they now have programmes to develop the technology. The optimism springs from the emergence of technology that promises to kick stem cell science into the fast lane, together with the collective knowledge of cell biologists who have been studying mouse ESCs for nearly two decades. One of the trickiest problems, for example, is how to keep stem cells dividing in a flask while preventing them from indiscriminately taking on specialfsed roles. Outside the embryo, ESCs try to re-enact development by forming "embryoid bodies", a nasty mess of tissues similar to that in teratocarcinomas. Researchers found they could sweet-talk mouse ESCs into dividing without transforming by growing them in the company of other embryo cells called fibroblasts. Gearhart and Thomson found the same trick worked for human ESCS. And while, even in the mouse, most of the developmental cues are still a mystery, some are now known. Retinoic acid, for example, will transform mouse ESCs into neurons. Other growth factors, such as BMP4, will persuade them to become muscle, bone and tendon cells. While these lab processes are grossly inefficient, the few cells that make the transition seem to be healthy. Injections of neurons created from teratocarcinoma cells, for instance, improve the cognitive and motor functions of rats with brain damage. The same test-tube neurons are now being used to treat human stroke patients in a small trial run by neurologist Douglas Kondziolka and his colleagues at the 'University of Pittsburgh Medical Center in Pennsylvania. Past work has shown that these neurons stop dividing, so they are unlikely to form tumours again. But since teratocarcinoma cells have genetic mutation, it would obviously be better to make the nerve cells from ESCS. Despite the successes two decades of struggling with mouse ESCs and teratocarcinoma cells, researchers are still nowhere near growing tissues at will. "At this point we're like tourists slowly shouting English at the natives," says Hogan.

Superhuman Slave

Today, about a hundred different growth factors have been identified, and more are being found all the time. It's likely that specific tissues will develop only when the correct combinations of these proteins are applied in the correct orders -and-that still leaves out the largely mysterious contributions made by physical forces. So, working out the recipes for each tissue type is still going to be a superhuman undertaking. One obvious way to pick out these recipes is to program a robot to slavishly try combinations of growth factors in every possible order. This is exactly what Joel Greenberger and Ray Houck have done at the University of Pittsburgh Medical Center. Greenberger wanted to find the magic combination of growth factors that would persuade haematopoetic stem cells, descendants of ESCs that spawn blood and immune cells, to carry on dividing in their unspecialised state. Large-scale production of these cells is a long-standing goal because they are used to treat blood and inunune diseases including leukaemia and severe combined immunodeficiency. Greenberger and Houck's combinatorial cell culture system is stocked with plates the size of a postcard, each containing around 1500 wells. Initially, each well has one stem cell. Then, once an hour, as the cells grow and divide, a robot arm moves each well to a n-dcroscope where its snapshot is taken and stored in a computer. Image analysis software combined with automated sampling of the cells, monitors how the cells grow, change shape and move, as well as the appearance of proteins on their surface, and which genes are turned on and off-all clues to how the cells are changing or keeping their identities. This cyberassistant has already revealed the growth factors needed to make haematopo(tic stem cells pass through several cycles of division without becoming specialised. Now the researchers plan to cash in on its talents by selling the machine to ESC biologists. A radically different approach is to allow ESCs to differentiate indiscriminately, and then find a smart way to pick out the few cells that have the characteristics you're after. With this in mind, molecular biologist Loren Field at Indiana University has fitted mouse ESCs with a gene for drug resistance connected to a genetic switch that is thrown only in heart muscle cells. Once the ESCs have differentiated into their usual smorgasbord of tissues, he simply adds the relevant drug and kills off everything but the heart muscle cells. When he injects these heart cells into mice, they integrate into the heart tissue, even beating in synchrony with the body's own cells (journal of Clinical Investigation, vol 98, p 216). So long as you know which genes are switched on in specific tissues-and such information is flooding out of genomics labs around the world-this selection should work with any type of cell, says Field. Genetic selection also has the advantage of automatically killing off undifferentiated ESCs which could form teratocarcinomas ff they were accidentally injected into a patient along with the specialised cells.


But the problem dogging all the techniques to date is that they are too much whip and not enough carrot. More cells die or ignore the signals than are converted to their new role. "What you end up with is a great big mess," says Peter Rathjen of the University of Adelaide in Australia. Even ff a few of the desired cells can be plucked out, they would have to be grown for many generations to make enough for a transplant. That prematurely ages the tissue, and in any case some specialised cells simply refuse to divide. If ESCs are ever goin to be widely used for @ 9 treating patients, biologists need to figure out how to convert them en masse into the target tissue. And in February's Journal of Cell Biology (vol 112, p 601), Rathjen reported the first glimmer of hope that this wfll be possible. His team has found the growth factor combination needed to convert ESCs in a flask into primitive ectoderm. "The cells seem ready for this change and not for any other,' says Rathjen. Intriguingly, with another switch of growth factors, the cells can also regress to ESCs. Vescovi suspects that a similar developmental reversion explains the startling metamorphosis of brain cells to blood cells. Growth factors in the body turned the neural stem cells back into ESCs, which then followed the developmental pathway that leads to blood cells. An explanation, says Weissman, is that ESCs are simply less ephemeral than everyone has assumed. "We thought such cells disappeared during development, but a few could be hiding in every adult tissue," he suggests. Either way, the ability of cells taken from an adult animal to transform from one tissue type into another raises the tantalising possibility of harvesting or creating cells that are just as powerful as ESCs from anyone's body That would not only silence critics worried about the ethics of using embryonic tissues for therapy, but would also eliminate any problems of tissue rejection (see 'A question of acceptability", p 35). So the real prize might not be ESCs themselves but the insight they give as to why some cells are trapped with their fate while others can switch identities. "If we understand what their genes are doing, we might understand the source of their power," says Gearhart. "It's a quest we're fuming our attention to.".. 11

Gone with the wind Will buffer zones stop genes spreading to nearby crops?
NS 17 Apr 99

POLLEN blown from large fields of genetically modified oilseed rape remains fertile over greater distances than expected, say British botanists. Their results could prompt a review of rules goveming flie size of the "buffer" zones between transgenic and natural plants-as the pollen fertilised plants after travelling twice the current buffer distance demanded in Britain. Environmentalists have wamed of dire consequences if genes that make crops resistant to herbicides spread to weeds. And organic farmers fear losing their status if their crops are pollinated by nearby transgenic plants. Such worries have already provoked one court case in Britain (This Week, 1 August 1998, p 5). To see how far live pollen could spread, Jeremy Sweet and Euan Simpson of the National Institute of Agricultural Botany in Cambridge studied a 9-hectare plot of transgenic oilseed rape. The rape was resistant to glufosinate, a weedkiller made by AgrEvo of Frankfurt. At various distances from the plot, the investigators grew male-sterile rape plants. Oilse6d rape frequently self-fertilises, but because they make no pollen themselves, male-7sterile plants are much more likely to be fertilised by airbome pollen. "There's no competition, so they accept anything that's flying around," says Simpson.

Afterwards, the researchers screened seeds produced by the male-sterile plants to see what proportion were resistant to glufosinate. Any resistant seeds must have been produced by plants crossfertilised by genetically modified pollen blown on the wind. Simpson and Sweet presented their results this week at a conference on gene flow and agriculture at the University of Keele in Staffordshire. Even at sites 400 metres away from the transgenic plots, as many as 7 per cent of the seeds were herbicide resistant. 'That is quite high," says Simpson. At 100 metres, between 8 and 28 per cent of the seeds were resistant. However, Simpson stresses that by using male-sterile plants, he and Sweet have examined a "worst-case scenario". "Research has shown that you can get significant cross pollination at up to 50 metres, but there ought not to be anything to worry about beyond that," say Simpson. According to the existing British rules, experimental plots of transgenic oilseed rape must be grown at least 200 metres from urtmodified crops. In earlier experiments, he and his colleagues examined 8000 seeds from normal rape plants growing up to 150 metres from a field of transgenic rape, but found none with seeds that became herbicide resistant through cross-pollination. "Fertile plants are a lot less lik-tly to be fertilised by incoming pollen," says Simpson. This year, the researchers will repeat their experiments replacing half of the male-sterile plants with normal plants. "We're putting both out so we can get a comparison," says Simpson. Andy Coghlan

Proteins on tap
NS 8 May 99

SQUEEZING human proteins out of plants could soon be easier than tapping latex from rubber trees, say biotechnologists who have learnt to make the roots of tobacco plants express human genes. Many human proteins, such as antibodies, are already produced in cultures of plant cells. But these cultures require an expensive sterile medium and it is difficult to extract the proteins from inside the morass of cells. Ilya Raskin at Rutgers University in New Jersey wondered if it might be cheaper and more practical to make plants secrete the proteins from their roots as they grow. Plant roots secrete many chemicals to alter the soil around them to make it to their liking and for self-defence. "They can only survive if they launch a continuous chemical attack on their environment," says Raskin. He and his colleagues took genes from humans, jellyfish and bacteria and added a sequence for a protein that plant roots secrete naturally. They inserted this gene into tobacco plant cells, which they then cultured in hydroponic solution so that they grew into mature plants. As the team reports in the current Nature Biotechnology (vol 17, p 466), the roots exuded large quantities of the engineered proteins into the solution. And although many proteins engineered into cells of other organisms are inactivated by foreign enzymes that alter them, these proteins functioned normally. The roots secrete the proteins throughout the plant's life and can be harvested from the growth medium. Roots also grow at high densities, so the proteins quickly saturate the hydroponic solution. "You are operating more like producing maple syrup or rubber," says Raskin. "if you had to cut down a maple tree to get syrup, it would be very expensive." Raskin and Photosynthetic Harvest, a company he helped found, are now trying to produce hepatitis B antigens for human vaccines in the tobacco root system. They hope to achieve full-scale production within a year. Jonathan Knight

Florigene's Moonshadow: a "blue gene" from petunias gives the carnation its startling hue

Blooming unnatural
NS 22 May 99
These flowers are unlike anything you've ever seen before

VIOLET carnations produced by genetic engineering will go on sale in the US and Europe later this year. The blooms, created by Florigene of Melbourne, have a gene from petunias that makes them violet, a hue they cannot produce naturally. The move seems certain to dismay envirorunentalists opposed to genetic modification. Founded a decade ago, Florigene has yet to realise its initial aim of breeding a blue rose. But through genetic engineering, it has produced several flowers with novel bluishviolet hues that are impossible to achieve through conventional plant hybridisation. "They go from mauve through to black, and none of these colours has existed before in carnations," says Peter Molloy, the chief executive of Florigene. "We have effectively created new species of carnations." Florigene is to launch the violet camation, which it has named Moonshadow, at a major horticultural show in Kansas City next month. In November, the plant will be launched in Europe at a global flower convention in Aalsmeer, the Netherlands. The plants will be grown under glass in Spain. Molloy believes the carnations pose no environmental threat-and the regulators agree. Florigene has approval to sell the flowers from the US and the European Commission. "The application to sell the plants went through unopposed," says a spokesman for Britain's Department of the Environment, Transport and the Regions. Molloy points out that the growing carnations will be confined in greenhouses, so no genetic material should escape. Florigene says that cultivated carnations produce relatively little pollen, and what pollen there is cannot be spread by the wind as it is heavy, sticky and buried deep within the flower. The flowers produce no new pollen after they have been cut. John Beringer, the chairman of Britain's Advisory Committee on Releases to the Environment, says that the cut carnations are essentially infertile and have no weedy relatives to which the violet trait could spread. Nor would weeds become more problematic if they became violet. "These are glasshouse crops that pose no threat to the environment," he says. Florigene has successfully test-marketed the new blooms in Australia. Next year, it will launch a black camation and a range of ordinary-looking camations genetically modified to last a month in the vase. The company discovered and patented the "blue gene" from petunias in 1991. The gene codes for an enzyme called flavonoid 3',5'-hydroxylase, which is vital for the creation of delphinidin, the blue pigment in a wide range of flowers including petunias, violets, hyacinths and irises. However, a true blue rose remains elusive, as delphinidin can be masked by existing pigments or chemically altered by high acidities in the petals, diluting its blue colour (see "Brave new rose", New Scientist, 31 October 1998, p 30). Molloy hopes true blue roses will emerge through crosses of the engineered varieties with native Japanese roses. Andy Coghlan

Worn away NS 29 May 99

THE ends of Dolly the cloned sheep's chromosomes are shorter than those of normal sheep of the same age. This suggests that she inherited some of the wear and tear suffered by her six-year-old mother's cells. Whenever a cell divides, the end of its chromosomes, called telomeres, erode away. When they wear down to a critical length, the cell stops dividing. Older animals have shorter telomeres, and biologists consider telomere shortening a hallmark of ageing. So when a team at the Roslin Institute near Edinburgh turned an adult cell Into Dolly, scientists wanted to know about her telomeres. Now the Roslin team and the affiliated company PPL Therapeutics say that Dolly's telomeres are shorter than those of two sheep cloned from embryonic cells-and all of the clones' telomeres are shorter than those of normal sheep (Nature, vol 399, p 316). For the time being, Dolly seems healthy. And biologists will need to study many cloned animals before they know whether their shortened telomeres will make them age more quickly. The problem could get worse if cloning is carried out over several generations. But normal reproduction seems to repair the shortened telomeres: Dolly's first daughter, a lamb called Bonnie conceived in the traditional way, has normal chromosomes. Given the uncertainties, however, Alan Colman of PPL Therapeutics says the findings provide "another good piece of evidence to make people shy away from human cloning". Noli Boyce

Male clone strikes a blow for equality
NS 5 Jun 99
UNTIL now, all the animals cloned from adult cells and described in the scientific literature have been females. That's why Fibrio, a mouse created from a cell from the tall of an adult male mouse, is cloning's latest celebrity. Fibrio, born last October, is unveiled to the world in this month's issue of Nature Genetics (vol 22, p 127). Teruhiko Wakayama and Ryuzo Yanagimachl at the University of Hawaii in Honolulu, who cloned female mice last year ("The year in news", 19 December, 1998, p 28), took cells called fibroblasts from the tips of the tails of IO-week-old golden-brown male mice. They injected nuclei from these cells into eggs which had been stripped of their own chromosomes. The eggs came from females with black coats. A third of the 700 embryos survived to be implanted, but most then failed to establish pregnancies. Of the three pups eventually delivered by caesarean section, two stopped breathing immediately. The lone survivor had a golden-brown coat just like his "parent". Although Fibrio is the first male cloned from an adult to be described in a scientific paper, he may not be the first one created. At a conferance in Hawaii in February, two Japanese groups claimed to have cloned bulls, and one team leader, Chikara Kubota of the CatUe Bm-eding and Development Laboratory in Kagoshima, told New Scientist that four clones made from skin cells of a single bull were bom last August. Experts say the main reason males haven't been cloned before is that many of the companies backing the work are interested in cloning female animals that could be genetically engineered to produce useful proteins In their milk. "There's no reason to believe that male cloning is harder," says Eric Overstr8m of Tufts University in North Grafton, Massachusetts, who helped a company called Genzyme Transgenics clone female goats engineered to make an anticoagulant (This Week, I May, p 5). r Jonathan Knight, San Francisco

What makes a human?
NS 26 Jun 99
That's the question at the core of an unusual patent application

CREATURES created from a mixture of human and animal cells cannot be patented, says the US Patent and Trademark Office. The ruling marks the beginning of a legal battle that opponents of biotechnology hope will undermine thousands of patents on organisms carrying human genes. Stuart Newman, a developmental biologist at New York Medical College, and antibiotech activist Jeremy Rifkin applied for the patent last year. Their intention was to use it as a test case to question the validity of other patents. "I have problems with thinking of life as intellectual property," says Rifkin, who runs the Foundation on Economic Trends in Washington DC. The part-human embryos mentioned in the patent have not been createdbut neither are they complete science fiction. Scientists have mixed cells from goat and sheep embryos to create viable "chimeras" containing a mixture of cells from both species. In their patent, Newman and Rifkin propose similar experiments, mixing various human cells with the embryos of chimpanzees, pigs or other animals, They suggest that such embryos might prove useful for research or have medical applications. The patent office rejected the application because it "embraces a human being". Ownership of such a patent could be seen as violating the 13th amendment to the US Constitution, which prohibits slavery. But the applicants argue that their claims are not so different from those made in thousands of other patents that use human materials. If an embryo with one human cell "embraces" a human being , then why doesn't a bacterium, sheep or cow that contains a human gene? If his patent isn't valid, claims Rifkin, neither are thousands of others that underpin the emerging field of molecular medicine. In particular, he points to a patent application made by scientists at Advanced Cell Technology (ACT) in Worcester, Massachusetts, who have created embryos by fusing human cells with cow eggs stripped of their chromosomes (This Week, 11 July 1998, p 4). ACT's project, which aims to grow cloned cells and tissues for transplant, continues to make headlines around the world-with some commentators alarmed about the project's perceived assault on the unique identity of the human species. To pursue their argument, Rifkin and Newman are appealing against the patent office's decision, and hope eventually to obtain a definitive ruling on the patent's legality from the US Supreme Court. Legal experts are divided. John Barton of Stanford University School of Law in California says the patent illustrates that there is no legal definition stating how much human material an organism must contain before it is considered human. "It's an uncertain line and it is going to have to be drawn at one point," he says. But Rebecca Eisenberg, a patent expert at the University of Michigan Law School in Ann Arbor, argues that patents are supposed to foster innovation, rather than being used to make wider legal and political points. "It's bogus and I won't be surprised if the courts simply refuse to hear it," she says. Philip Cohen

Healing honey
NS 26 Jun 99
Flowers are being turned into vaccine factories

A SPOONFUL of honey could help the medicine go down, say Dutch biologists. They are genetically engineering plants so that honey made from their nectar will contain drugs or vaccines. The honey could either be fed directly to patients, or drugs could be extracted from it. "It's a production system that would require very little purification," says Tineke Creemers of the Centre for Plant Breeding and Reproduction Research in Wageningen. "The protein is concentrated by the bees, so it's a very cheap production method." The researchers also hope that the sugars in honey will act as a preservative, and are investigating whether proteins in honey retain their activity even if it is not refrigerated. If so, this would be a boon for vaccination programmes in poor tropical countries, which are often hampered by shortages of cooling equipment. Two discoveries came together to spawn the project. First, to their surprise, Creemers and her colleagues discovered antifungal proteins in nectar from common heather, Caluna vulgaris. They wondered if bees pass the proteins undigested into honeyand tests of commercial brands showed that they do. The researchers also fed bees a sugar solution laced with a protein called bovine serum albumin. "The proteins remained intact in the honey and were concentrated twofold compared with the original solution," says Creemers. Secondly, she and her colleagues discovered a genetic switch, or promoter, which activates genes in the nectary, the organ in plants where nectar is made. So they decided to try to add genes for various drugs to plants in such a way that the genes would be activated by the nectary promoter. Because the promoter is specific to the nectary, these drugs should be produced only in the nectar, where bees could eat them. They are in the process of genetically engineering petunias so that they produce a vaccine against a disease of dogs caused by a parvovirus. The active component is part of a surface protein made by the virus, which should trigger immunity in dogs. "The dogs would either eat the honey as an oral vaccine, or the vaccine would be purified and injected," says Creemers. Once the plants are fully grown and begin producing nectar, bees will be unleashed on them to produce honey that the researchers hope will contain the vaccine. Creemers and her colleagues expect to harvest the first honey in a year's time. "It's an exciting variation on making vaccines in plants," says Charlie Arntzen of Cornell University in Ithaca, New York, who is producing bananas engineered to contain vaccines. Creemers and her colleagues are doing their experiments in glasshouses, to ensure that their bees feed only on the modified plants and to minimise concerns of the vaccine genes being spread by pollen. Because of this they are using bumblebees, which are easier to manage in a contained environment than large colonies of honeybees. Andy Coghlan

Out of Egypt A powerful organic pesticide could end up in modified crops
NS 19 Jun 99
ORGANIC farmers and biotech firms alike stand to benefit from a potent biopesticide that kills a multitude of insect pests without harming beneficial species. The biopesticide, a strain of Bacillus thuringiensis, produces more toxins than any other known strain of the bacterium. The supercharged B. thuringiensis was discovered by Egyptian researchers in cadavers of larvae from the pink bollworm, which ravages cotton plantations. The researchers have already sequenced and patented some of the key genes that make the toxins, and hope to license a seed company to use them in genetically modified crops. Farmers have been using strains of B. thuringiensis as organic biopesticides for three decades. But whereas existing commercial strains make just one or two Bt toxins that kill the pests, the Egyptian variant makes a staggering 18. "We were amazed and very happy when we stumbled on it," says Yehia Osman, head of the team which isolated the bacterium at the Agricultural Genetic Engineering Research Institute in Giza, Egypt. "This is the most potent Bt strain yet found, and has the most diversified host range." So far, Qsman and his colleagues have discovered that the toxic proteins kill moths, coleopteran beetles and dipteran insects such as mosquitoes. As an added bonus, the toxins also kill nematode worms. "It's an ideal to have a single isolate with this much activity," says Osman, who presented his findings earlier this month at the annual meeting of the American Society for Microbiology in Chicago. "The insects would have a hard time developing resistance to this." But recent reports have called into question the safety of existing commercial Bt strains. French researchers found that mice contracted fatal lung infections after inhaling the bacteria (This Week, 29 May, p 4). And at the Chicago meeting, Azam Tayabali of Health Canada, the Canadian health ministry, revealed the results of a study which showed that bacterial spores in some commercial Bt strains can kill human cells. He recommended that farmers spraying Bt mixtures wear proper protective gear. But Osman and his colleagues insist their tests indicate that the Bt strains they are using will prove safe for organic farmers to apply as a biopesticide: "We've done toxicity tests in rats and fish, and didn't get any adverse effects at all," Osman told New Scientist. "And so far, during all tests, it doesn't have effects on non-target organisms." The Egyptian Ministry of Agriculture is now undertaking trials. Osman's team has worked out the DNA sequences of several of the genes producing the toxins. The institute has patented the commercial application of the bacterium, and has an agreement with the Iowa seed company Pioneer Hi-Bred to potentially license use of the genes in genetically modified crops. Andy Coghlan

Safe havens
NS 19 Jun 99
Insect refuges may be set up near genetically modified crops

FACED with fears that some genetically engineered crops could render a popular natural insecticide useless, the US Environmental Protection Agency has proposed tough new planting rules. The EPA rules would require farmers to plant "reserves" of non-engineered crops close to those modified to carry genes for natural insecticides called Bt toxins. The genes come from the soil bacterium Bacillus thuringiensis, which is spayed on crops to kill insect pests. But scientists have pointed out that pests are more likely to become resistant if they are continually exposed to the toxins in modified plants. Environmentalists, government and industry have agreed that this problem could be solved by growing reserves of non-engineered crops. The reasoning is that the pests in the reserves would never build up a resistance to the toxins. So even if insects in the engineered fields do become resistant, the two pest populations should mix so that their offspring have little or no tolerance. How large and how close to the modified crops the reserves should be, however, has been hotly debated. The National Com Growers Association, along with major seed companies such as Monsanto and Novartis, proposed that 20 per cent of any maize crop should be a non-modified refuge, and that the refuges be grown within 800 metres of the modified plants. But last week the EPA suggested that 40 per cent of maize crops should be non-modified, and that these reserves should be planted within 500 metres of the modified plots. The rules would also lay down refuge requirements for modified cotton and potatoes. "I am pretty impressed," says Jane Rissler, a scientist with the Union of Concemed Scientists (UCS), a pressure group in Washington DC. "They have come a long way." However, even the EPXs proposal falls short of the 50 per cent non-modified quota favoured by the UCS, following recommendations made in 1998 by a group of six experts on Bt resistance. An expert panel is discussing the EPA proposals at a public meeting in Washington DC this week, but a final decision is probably months away Kurt Kleiner

The gentle touch
NS 11 Sept 99
Why replace broken genes when you can fix them?

A MORE subtle approach to gene therapy could provide a permanent cure for some genetic diseases. The first human trials, on children in the Amish religious community suffering from a rare syndrome called Crigler-Najjar, may begin next year. Conventional gene therapy relies on modified viruses to get healthy genes into cells. But the genes are inserted randomly into the genome, which may disrupt other genes. It also means that the activity of the introduced genes is unlikely to be regulated in the same way as it would be in a normal, healthy cell. Given these problems, some researchers have been trying to repair damaged genes rather than adding healthy ones. The best they had managed was to correct I per cent of cells cultured in a Petri dish (New Scientist, 11 April 1998, p 7). Now Clifford Steer and his colleagues at the University of Minnesota Medical School in Minneapolis have achieved much better results in rats, using a technique that exploits the body's own DNA repair mechanisms. The rats were missing a single nucleotide in the gene for a liver enzyme that breaks down bilirubin, a toxic waste product created when the body destroys old red blood cells. Steer and his colleagues injected them with microscopic fat globules containing "chimeric" molecules made of DNA and RNA. These were designed to bind to the target DNA in such a way that DNA repair enzymes would make the change that fixes the fault. The same technique has been used to genetically engineer plants (Nezt? Scientist, 31 July, p 4). A single injection into the rats' tails led to a significant decrease in bilirubin levels that seemed to be permanent. in the latest Proceedings of the National Academy of Sciences (vol 96, p 10 349), Steer reports that the genetic defect was fixed in up to 20 per cent of the rats' liver cells. "This is the first time anyone has been able to go in and correct a gene defect in an animal," he says. A similar genetic defect causes CriglerNajjar, which is unusually prevalent in the Amish of Lancaster County, Pennsylvania. Children who have the syndrome are required to sleep under special blue lights that destroy bilirubin-a practice tolerated by the Amish despite their reputation for shunning technology. If this fails they may need a liver transplant. Steer hopes that if his gene-repair technique works in people as it does in rats, it may be possible to cure Crigler-Najjar. Other gene therapists are excited by Steer's rat results-particularly since conventional gene therapy doesn't usually alter such a high proportion of the target cells. Mark Kay of Stanford University in California admits he was sceptical about results that Steer published last year. "But I don't think there's any doubt any more." The limitation of the technique is that it can only change or add one nucleotide at a time. "Sickle cell and cystic fibrosis are two very good examples of where this would be useful," says Kay. But many genetic diseases involve more extensive defects. Nell Boyce, Washington DC

NZ Herald Monday, May 29, 2ooo

GE genes can jump species: research

LONDON Research by a leading German zoologist has shown that genes used to genetically engineer crops can jump the species barrier. A three-year study by Professor Hans-Heinrich Kaatz at the University of Jena found that the gene used to modify oil-seed rape had transferred to bacteria living inside honey bees. The fuidings, reported in British newspapers, will undermine claims by the biotech industry and supporters of GE foods that genes cannot spread. They WW also increase pressure on farmers across Europe to destroy fields of oil-seed rape contaminated with GM seeds. Kaatz told the Observer. 'I have found the herbicide-resistant genes in the rape seed transferred across to the bacteria and yeast inside the intestines of young bees. This happened rarely, but it did happen." Asked if his flndings had implications for the bacteria inside the human gut, Kaatz replied: 'Maybe, but I am not an expert on this.11 The Obsemer said Kaatz was reluctant to talk about his work until it was officially published and reviewed by fellow scientists. The reports come a day after BriLiin's Agriculture Minister, Nick Brown, urged farmers to destroy crops contaminated with GE seeds. AFP