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Mice GM with jellyfish genes glow in the dark NS 8 May 1999

DNA Chips

New Scientist 11 Mar 2000

WILL "DNA chips" that reveal your genetic makeup within minutes prove to be aivesome medical tools or the means of genetic discrimination? We could find out sooner than anyone expected. A British biotech start-up has filed for a patent on a device that can detect variants of over 2500 genes-including genes that affect behaviour and intelligence. Researchers with the company Genostic Pharma of Cambridge have worked out the blueprint for a system which, they believe, can provide a "core' genetic profile of any individual. The automated device uses DNA chips-essentially sensors that can detect many thousands of gene fragments at once. The company expects to start making prototype chips within months. it says that the device will help doctors to find out if people are predisposed to particular diseases and tailoring treatments to individuals. But such devices could also be used by unscrupulous employers or insurance companies to reject applicants with "the wrong genes". Rival DNA chip systems tend to focus on gene variations relevant to just one disease, such as breast cancer, or to a potential adverse drug reaction. But Genostic Pharma's chip gives medically relevant genetic information about 16 different types of disease (see Figure), says the company's founder Gareth Roberts. Roberts and his colleagues at Genostic Pharma combed the scientific literature for common gene mutations called singlenucleotide polymorphisms, or SNPS, which can alter people's predisposition to disease or their reaction to drugs (New Scientist, 14 November 1998, p 32). They then plucked the entire sequences for these genes out of publicly available sequence databases. In a 700-page patent application, Roberts lists all the chosen genes plus known variants. To create its genetic profiling system, Genostic Pharma must take short stretches of DNA from each variant of each gene and fix them to a specific spot on the surface of the DNA chip. To test a blood or saliva sample, researchers will break down its DNA into fragments, attach fluorescent markers and wash them over the chip. The fixed DNA strands will grab any matching gene fragments that pass by. The fluorescent tags will then reveal by their position the identity of each gene captured from the sample (New Scientist, 14 November 1998, p 46).

The genes Roberts has chosen to include on the profiling chip are the ones he thinks are relevant to a range of conditions, from cancer and heart disease to headaches and impotence. But others doubt enough is yet known about the human genome for Roberts to make these choices. "Where Genostic comes up with thousands of gene variants to put on their chip is a mystery to me," says Francis Collins, director of the US National Human Genome Research Institute near Washington DC. Daniel Cohen, chief genomics officer at Genset of Paris, agrees. 'There haven't been enough populafion studies, as far as I know, to assess with enough precision the risk or predisposition for any of the diseases mentioned in the patent," he says. Others are not convinced that the chip is intended for entirely therapeutic purposes. The patent lists 500 variants of genes related to psychoses and personality, for example, as well as 250 linked to behaviour. "It creates a set of issues way beyond medical applications," says Onora O'Neill, former chair of Britain's Human Genetics Advisory Commission. Andy Coghlan

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Keep that spray NS 18 Dec 1999

Crops made resistant to pests still do better with chemicals

FARMERS may need to douse their fields with yet more pesticides to get the best out of genetically modified plants. At least, that's the implication of patent applications filed by Novartis of Basle in Switzerland, one of the leading companies in the field. The applications (WO 99/35910 and WO 99 / 35913) were filed after scientists at Novartis realised that a wide spectrum of insect pests was attacking Bt maize, its major GM crop. Genes inserted into the maize enable it to make the Bt toxin, a bacterial protein that kills European com borer larvae. These larvae chew their way into the stems of young maize plants and can kill them before they get established. But many GM plants that saw off the borer larvae were later attacked by sapsucking insects. "Bt toxin has a rather narrow spectrum of activity, so you don't get control of all pests," says Walter Smolders, head of patents at Novartis Seeds. To find a way round the problem, Novartis scientists tried applying different combinations of the company's pesticides to the Bt maize. Their patents identify combinations of pesticides that could raise yields of the maize by 20 per cent. The same pesticides appear to increase the yields of other GM plants, including those engineered to resist the effects of herbicides. So Novartis has extended its patents to cover use of the pesticides on a long list of transgenic crops including maize, cereals, soya beans, potatoes, rice, cotton and mustard. If the patents are granted, this means they will also apply to crops from competitors such as Monsanto of St Louis, Missouri. Heinz Hammann, head of patents in Novartis's crop protection division, claims the pesticides mentioned in the patents are mostly environmentally benign, killing only the pests which attack the plants. Maize, for example, is vulnerable to sapsuckers such as the flea beetle (Phyllotreta agriotes) and various aphids. "Non-target species don't suck the plants, so they're not harmed," he says. But some of the pesticides are less friendly. Carbamates, for example, act on the nervous system of pests and are known to affect birds, fish, game, bees, mammals and other farmland wildlife. And given that agribiotech firms have consistently argued that GM crops will reduce pesticide use, Novartis's patent applications are sure to be seized upon by groups that oppose the technology. Brian Johnson, head of the biotechnology advisory unit at English Nature, a conservation watchdog, says he wants to see evidence confirming Novartis's suggestion that the use of pesticides on GM crops outlined in its patent applications will be less environmentally damaging than conventional chemical treatment of ordinary maize. "It's the impact of the whole process on biodiversity that counts," he says. "But the impacts of what they are proposing are not known." Andy Coghlan and Barry Fox

NS15 Jan 2000\

Missing the target: Gene therapy's most promising weapons may have a limited range

PLANS for using HIV and related viruses to overcome a major hurdle in gene therapy have been dealt a blow. Unlike most of the vectors used in gene therapy, HIV and other "lentiviruses" should be able to deliver genes to cells that aren't dividing. But the latest research shows that they don't always do so. Many gene therapy trials have yielded disappointing results, mainly because so few cells in the target tissue take up the therapeutic gene. At any one time, the vast majority of our cells aren't dividing. So finding a vector that can carry genes into these cells should help overcome this problem-and several experiments with lentiviruses have suggested that they just might do the job. Following early successes, several groups are now planning to use HIV, stripped of the genes that make it so deadly, for gene therapy against AIDS (New Scientist, 6 February 1999, p 5). And other research teams are experimenting with similarly genetically disabled lentiviruses, such as equine infectious anaemia virus, for a wide range of gene therapies. But Mark Kay, Frank Park and their colleagues at the Stanford University School of MedicinEr in Califomia have now shown that lentiviruses can fail to deliver. They loaded harmless variants of HIV with a marker gene and injected them into mice through the vein from the gut that enters the liver. V"en the researchers later examined the organs, they found that the gene

was only taken up by those rare liver cells that were dividing or were in the process of copying their DNA-A necessary prelude to cell division. Worse still, Kay and his colleagues found that when the lentiviruses were given in higher doses, they appeared to cause liver damage. Large amounts of the enzyme alanine aminotransferase, which is released by damaged liver cells, showed up in the animals'bloodstreams. The results are bad news for gene therapists who are backing the lentivirus approach, as the liver is the main target for attempts to introduce genes to correct a wide variety of metabolic disorders. But lentivirus enthusiasts say that results in other tissues are still looking good. "The liver is only one target organ," says Sue Kingsman, scientific director of Oxford BioMedica, a British company developing lentiviral gene therapies to fight cancer. 'We've done a lot of work with lentiviruses in the mouse brain, and they've been fantastic, with high doses and no signs of toxicity," she says. Experiments with human cell cultures are also yielding encouraging results, says Kingsman. "With the equine virus, time and time again we get good transfer in human cells that don't divide." "It's not the end of the line," agrees Kay, "but it narrows the applications to some degree. Lentiviruses may not be as broadly useful as originally thought." Andy Coghlan

Source: Nature Genetics (vol 24, p 49)

Clones may not grow old before their time

SIX calves from Connecticut, cloning's latest stars, should finally answer the question of whether clones age prematurely. Dolly the sheep, the first animal to be cloned from an adult cell, appears healthy so far. But there are signs that the wear and tear accumulated by that donor cell could make her age before her time: Dolly's chromosomes are shorter than normal sheep of the same age, which is a sign of cellular ageing (New Scientist, 29 May 1999, p 12). Even if Dolly dies young, however, it might just be that longevity doesn't run in her family. Since Dolly's relatives aren't around, Jerry Yang at the University of Connecticut in Storrs and his colleagues are studying the way clones age more precisely. To see how cellular ageing might affect ageing of animals cloned from the cells, they took cells from the ear of a 17-year-old bull and allowed them to divide in culture for differing periods of time. Some cells divided five times, others as many as 30 times. "Each division is like ageing the cells about one year," says Yang. So by

comparing animals cloned from cells cultured for varying periods, it should be possible to tell if older cells yield clones that age faster. Because the breed of bull used by Yang typically lives to about 25 years old, he suspected that the oldest cells wouldn't be useful for cloning. But they actually seemed to work better. Only 30 per cent of embryos produced from cells that had divided 10 times established pregnancies, compared with 64 per cent for cells that had divided 20 times. And although the number of pregnancies that went to term was small, these tended to be from cells cultured for longer. "Getting healthy births from such old cells suggests that ageing might not be as big a concern as we thought," says Jim Robl of the University of Massachusetts at Amherst, who also works on cow cloning. Yang intends to monitor the calves, looking for signs of accelerated ageing such as chromosome shortening and declining immunity. "These bulls will be watched closely day and night the rest of their lives," he says. Philip Cohen

NS 30 Oct 99 Toxic block

Genetically engineered maize that produces the insecticide Bt is less prone to mould. Gary Munkvold of Iowa State University says caterpillars killed by Bt maize spread the spores of moulds and promote infection by wounding plants. Fusarium mould produces toxins called fumonisins, which poison cows and pigs and may be human carcinogens, while Aspergillus produces toxic aflatoxin. Munkvold reports that some Bt maize carries less than a tenth as much fumonisin and less than a quarter as much aflatoxin as non-Bt strains.

We have the power 23 Oct 1999

A safer way of altering genes will make engineering humans more tempting than ever

MICE engineered to carry an extra artificial chromosome have successfully passed it to their offspring. Although the Canadian company responsible has no intention of repeating the experiments in people, its work shows that human germline gene therapy-making genetic changes that will be inherited by future generations-is becoming a practical possibility. Chromos Molecular Systems of Bumaby, British Columbia, reported the breakthrough this week in London at a conference on biotechnology. "It's the first time an artificial chromosome has ever been shown to be inherited in any mammal," says Eileen Utterson, vice-president of corporate development. Chromos plans to use the technology to create herds of genetically modified animals whose milk will contain pharmaceuticals. Geneticists have been creating transgenic animals for years by injecting genes into a newly fertilised embryo. When this method works, every cell in the animal that develops from the embryo will contain the added genes, including its sperm and eggs. This means that the genes will be inherited by the transgenic animals' offspring. It's a haphazard process, however. Often the gene doesn't get incorporated into the embryo's genome. And when it is, the gene splices itself at random into one of the animal's chromosomes, where it may not work as normal or, worse still, can disrupt other genes-potentially causing developmental abnormalities.

While occasional "mistakes" are tolerated in animal experiments, the danger of causing congenital defects is one of the reasons why germline gene therapy for people has remained taboo. So instead of trying to correct genetic defects such as the mutations that cause cystic fibrosis at the start of life-by adding genes to embryos created by IVF-gene therapists treat people with genetic disorders by adding therapeutic genes to specific tissues in the hope that they will be taken up by enough cells to correct the defect. While this ensures that sperm and egg cells do not become contaminated by the added genes, it isn't very efficient. As a result, many gene therapy trials have had only limited success. However, if genes could be ferried into embryos in an artificial chromosome that would safely be inherited without interfering with the rest of the genome, germline gene therapy might not be so risky. Chromos's experiments with mice suggest that this should be possible. "Because the artificial chromosome is separate, it doesn't interfere with the cell's own genetic machinery," says Utterson.

In London this week, Chromos presented preliminary results of experiments with mice given an artificial chromosome. By taking cell samples and exposing them to fluorescent dyes that bind to different parts of the chromosome, Chromos's scientists were able to discover which animals had accepted the chromosome. When the mice carrying the extra chromosome were crossed with normal mice, it was inherited in exactly the same way as the animals' natural chromosomes. Chromos is also working on human artificial chromosomes for use in conventional, non-germline gene therapy. Artificial chromosomes will have an advantage here as well, because they can carry much more DNA than is possible with existing methods, which use viruses or loops of bacterial DNA known as plasmids. But the company says it won't let its technology be used for human germline engineering. "We are in control of the technology, and we don't want to engage in germline gene therapy," stresses Utterson. However, many groups worldwide are also striving to create artificial human chromosomes. And some geneticists are talking openly of one day using such chromosomes for germline gene therapy (New Scientist, 3 October 1998, p 24). "This is obviously going to open up the debate again in the field of germline gene therapy," says Norman Nevin of Belfast City Hospital, who chairs Britain's Gene Therapy Advisory Committee. However, scientists advising the world's governments remain cautious. Claudia Mickleson of the Massachusetts Institute of Technology, who chairs the US National Institutes of Health's Recombinant DNA Advisory Committee, says that her committee wouldn't approve a germline trial without extensive preclinical information on safety. And given concerns about the technology being used to create "designer babies", Mickleson also says trials would not proceed without "intense discussion" with the public. Andy Coghlan

Decision time
Ns 8 April 2000

A single protein may be the first step to customising human stem cells

THE discovery two years ago of a technique for creating an immortal line of human embryonic stem cells (ESCS) raised hopes that it would one day be possible to grow endless supplies of specialised cells, or even organs for transplants. But researchers are still puzzling over the exact biochemical signals needed to control stem cells. A key step on that road has now been taken with a study of mouse ESCS, which shows that different levels of a "gatekeeper" protein called Oct-3/4 steer the cells towards becoming placenta, body tissues or another generation of ESCS. "The Oct-3/4 work tells us we are going to figure all this out," says Ron McKay of the US National Institutes of Health. "This is a technology, not a stab in the dark," says McKay. ESCs "are going to become a central part of modern medicine". A mind-boggling array of protein signals turns ESCs into the specialised cells of the body. What's more, to get the millions of cells that would be needed for each transplant, researchers must find an easier way to grow human ESCS. At the moment, human ESCs will only multiply if they are mixed with mouse cells. They also have a tendency to go backwards in development, turning into trophectodermcells that become placenta. Understanding what makes ESCs multiply is the first stage in solving those problems. Austin Smith of the University of Edinburgh and Hitoshi Niwa of Osaka University in Japan suspected that Oct-3/4-a transcription factor protein, which regulates gene activity-might be involved. They genetically engineered mouse ESCs to produce different levels of Oct-3/4. Their results showed that a finely tuned amount of the protein locks mouse ESCs into a state where they multiply without becoming specialised. When the researchers tumed up the amount of Oct-3/4, the cells switched identities into mesoderm and primitive endoderm-tissues that spawn body cells and the embryo's yolk sac. When they tumed it down, the ESCs became trophectoderm. The team has licensed this technique to the biotech company Stem Cell Sciences in Australia. In a separate development, Martin Pera and his colleagues at Monash University in Melbourne and the National University of Singapore have become only the second team to establish an immortal line of human ESCS, and have followed the cells' differentiation into primitive muscle and nerve cells. The reason it has taken two years to replicate the original finding is due in part to regulations that limit human embryo research in many countries. Such research is legal in Singapore.

Pera's team also showed that human ESCs make Oct-3/4, which some researchers had doubted. Taken together, the findings hint at a possible reason for some of the problems with human ESC cultures: varying levels of Oct-3/4 may be stopping the ESCs from multiplying. "It's an absolutely key issue, particularly because human ESCs tend to differentiate into trophectoderm," says Smith. Rachel Nowak

Hold the radicchio NS 30 Oct 1999

Bioengineered salads are off the menu in Europe

EUROPEANS won't be eating genetically modified salad any time soon. Faced with widespread public suspicion of GM food, a Dutch seed company seeking approval for modified salad leaves in the European Union now says it won't market the products even if it is given permission to do so. The company, Bejo Zaden of Warmenhuizen in the Netherlands, is applying to the European Commission's Scientific Committee on Food for permission to sell GM green-hearted chicory and radicchio rosso seed to farmers. The plants are modified to be pollen-free, allowing Bejo to control pollination and produce high-yield hybrids. But any decision will make little difference to the plants' commercial prospects. "It is obvious that there must be public acceptance first," says Dick Vanderzeiden of Bejo. He predicts that it could take 15 years before Europeans accept raw GM foods. The GM food products approved in Europe so far are cooked and processed before being eaten. In a separate development last week, the EU agreed that processed foods containing no more than 1 per cent of GM material in any ingredient can be labelled as GM-free. Any modified DNA in processed foods is likely to be destroyed, but intact DNA from raw vegetables might be taken up by gut bacteria. This possibility alarmed British officials who reviewed Bejo's application. The salad leaves retain a marker gene that confers resistance to the antibiotic kanamycin.

Although this antibiotic isn't widely prescribed, bacteria acquiring this gene may also become resistant to streptomycin and spectinomycin. "We've turned it down," says.a spokeswoman for the British govemment. "It's for the Commission and the Dutch company to decide what to do next." The compan@ is confident that resistance to the other antibiotics won't be passed on. But one British expert, who asked not to be named, argues that Bejo should have removed the gene. "It's basically fairly lazy genetics," he says. The US Food and Drug Administration approved the radicchio rosso in 1997, though Bejo has yet to market it. But according to the Biotechnology Industry Organization in Washington DC, raw GM foods such as tomatoes, papayas and squashes are already on sale in the US. Maft Walker

On your markers NS 20 Nov 99

New ways of engineering plants could win over sceptics

WILL a spoonful of sugar help genetically modified food go down? Novartis clearly hopes so. The Swiss multinational has developed a sugar-based replacement for the controversial antibiotic resistance marker genes used in some GM foods. Because genetic engineering is a hit-andmiss affair, marker genes are used to reveal if cells have taken up packages of new genes. After adding the genes to plant cells, the cells are exposed to antibiotics. The uninodified cells die, leaving botanists with the live ones that have taken up the new genes. Some scientists fear that when people eat GM foods that contain such marker genes, they might spread to potentially harmful gut bacteria, making them resistant to antibiotics. Novartis says there is no evidence this has ever happened, and that marker genes in products such as its GM maize pose no risks. Earlier this year, however, tests in the Netherlands showed that DNA could survive in the intestine for several minutes, suggesting that marker genes could be transferred to bacteria (New Scientist, 30 January, p 4). And some countries have been reluctant to approve crops that contain such genes. So to allay such concerns, researchers at Novartis's molecular biology lab in Rayleigh, North Carolina, have developed a sugar-based alternative to antibiotic-resistance marker genes. "We've already transformed a dozen crops, including maize, wheat, rice, sugar beet, oilseed rape cotton and sunflowers,"

says Willy DeGreef, head of regulatory affairs at Novartis. "Several are already in field tests and, if all goes well, we hope to apply for commercial release of at least one of them as early as 2001," he says. Novartis's new marker gene, mana, enables plants to digest a simple sugar called mannose-6-phosphate. Most plants can't handle this sugar, and so die when fed mannose alone. The mana gene codes for an enzyme called phosphomannose isomerase, which converts mannose-6phosphate into fructose-6-phosphate, another sugar that all plants can digest. The system cannot work in all plants because some, such as soya and other legumes, already have the mana gene. 'But we are lucky because most higher plants don't have it," says DeGreef. The fact the gene already exists in many familiar crops should increase confidence in the safety of the system, he says. "It's in many of our foodstuffs and in most of the animals we eat, and all mammals including us," he says. It also exists in many species of gut bacteria. Novartis unveiled the new marker system this week in London at a conference organised by Nature Biotechnology. The British Medical Association, which opposes the use of antibiotic resistance markers in food, welcomes Novartis's development. "The industry is beginning to take seriously the expressed concerns of others," says Vivienne Nathanson, the BMA!s head of health policy. Andy Cogbian

Japanese cloning pioneers break the rules NS 20 Nov 99

AN ATTEMPT to repeat controversial experiments in which human cells were fused with cows' eggs has landed a team of Japanese researchers in serious trouble. The Ministry of Education is now investigating the project as a breach of its ethical guidelines on human cloning. The research was carried out a year ago by Setsuo lwasaki's team at the Tokyo University of Agriculture and Technology. Following in the footsteps of the company Advanced Cell Technology in Worcester, Massachusetts (New Scientist, 1 1 July 1998, p 4 and 21 November 1998, p 14), lwasaki removed the chromosomes from 27 cows' eggs, which he then fused with human cells-in this case cancerous blood cells cultured from leukaemia patients. When the Yomiuri newspaper ran the story of lwasaki's experiments last week, he told the paper that he hoped to isolate embryonic stem cells-from which all the body's tissues eventually develop. This would have meant culturing the hybrid embryo for about five days, until it formed a hollow ball of cells called a blastocyst. However, most of the embryos did not develop and none underwent more than three cycles of cell division. lwasaki's work has been greeted with widespread concern in Japan, where guidelines designed to prevent human cloning bar researchers in universities and other public research labs from fusing human cells with eggs. He had not cleared the experiment with his university believing the guidelines did not apply to cows' eggs. The university says that future proposals will be rigorously screened by a new ethics committee. Peter Hadfield, Tokyo

Toxic leak NS 99

INSECT-KILLING toxins from genetically modified maize plants leak into the soil and persist for weeks, biologists in the US have found. Neither finding was expected, say the researchers, raising questions about the impact of the toxins on soil ecology. Guenther Stotzky and Deepak Saxena of New York University grew a commer- cial maize variety that is genetically modified to make the Bt toxin, a protein normally made by the soil bacterium Bacillus thuringiensis. The toxin protects the plants against moth larvae that burrow into maize stems. Stotzky and Saxena, working with Saul Flores of the Venezuelan Scientific Research Institute in Caracas, grew the maize in the lab and collected soil extracts from around the roots of the plants. They found Bt toxin in the extracts, which could still kill larvae in standard tests of its activity after 25 days.

The toxin could have widespread effects on soil ecology, say Stotzky and Saxena, as Ot maize plants now cover 6 million hectares of farmland in the US. They speculate that its effects might be beneficial, in killing pests that would otherwise damage the roots of crops. But the toxin could also harm benign soil organisms, or select for pests resis- tant to it. "If the target organisms become resistant, the technology of using the toxin goes down the tubes," says Stotzky. Environmental groups have seized on the findings. "I think it's time to revisit the environmental risk assessment for GM maize," says Sue Mayer of Gene- Watch, an environmental lobby group in Buxton, Derbyshire. "The release of the Bt toxin raises serious questions about soil ecology." Andy

Rivals Clash over rights to our genes NS 30 Oct 1999

MOVES by a private company to secure patents on 6500 human genes have been condemned by the alliance of government and charity-funded labs leading the Human Genome Project. Celera Genomics of Rockville, Maryland, was brmed last year by veteran DNA sequencer Craig Venter. The company claims it has sequenced 1-2 billion DNA bases-one-third of the human genome-within the past month alone. And last week it announced that it is seeking patents on a wide range of genes, including those for hitherto unknown cell receptor molecules, ion channels and signalling molecules. Members of the Human Genome Project, including the Welicome Trust charity in Britain and the US National Institutes of Health, are furious that Venter appears to have broken his earlier promises not to patent human genes en masse (New Scientist, 23 May 1998, p 4). "It's what they said they were not going to do," says John Suiston, director of the Sanger Centre near Cambridge, which is coordinating the Wellcome Trust's sequencing effort. The trust has vowed to challenge in court any patents which it considers invalid or so wide-ranging that they would restrict research by other groups. However, Celera says that the 6500 applications are provisional, and that it may end up submitting fully fledged applications on as few as 200 genes. The company also admits that its sequences still need to be checked to eliminate overlaps and duplications. "We need to sequence 10 times to get an accurate picture says a spokesman. The official genome project made this a priority from the start. "In the public domain one quarter of the genome has been sequenced at good accuracy" says SulstonCoghlan