New Foods and New Factories

A Fish Story

In 1994 Robert Devlin, working at the Canadian Department of Fisheries and Oceans, inserted a fish growth hormone gene into the eggs of the Pacific coho salmon. After fertilization, about 6% of the resulting embryos took up the gene and developed into fish with double the natural supply of growth hormone. The results were astounding. The transgenic fish grew faster and bigger than their natural siblings. A few were truly giants, reaching a weight 30 times greater than the average of coho taken from the sea! Unlike people, fish grow throughout their life span, so their actual weight limits could be even greater. The transgenic fish also matured faster, which is crucially important to efficient food production. When interviewed after their research was published in the prestigious journal Nature, the scientists guardedly predicted a 20-year lead time to commercialization.

If Elliot Entis and his colleagues have their way, that prediction will turn out to be terribly wrong. They are fish farmers, not fishermen, fish farmers. And they have a fish story that trumps any of the yarns one might hear on the docks of Key West. The difference is that they have the pictures to prove it. Their company, A/F Protein, Inc., based in Waltham, Massachusetts, is committed to using transgenic techniques to change the way that the Atlantic salmon (as well as several other species, including trout), one of the most popular and expensive fish, reach your dinner table. The goal at A/F Protein is to grow much bigger salmon, much more quickly, in a much cleaner environment, and at a much lower cost. Since its scientists developed a transgenic approach to creating bigger salmon in the mid-1990s, the company has made impressive progress. At the end of their first year of life, its transgenic salmon are four to six times heavier than are one-year-old Atlantic salmon netted in the ocean or nontransgenic salmon grown under identical conditions. Laid side by side, the cultivated fish towers over the others, and virtually all the weight difference is due to muscle.

How were the transgenic fish created? The scientists at A/F Protein achieved these astounding results by inserting an additional copy of the salmon growth hormone gene into early salmon embryos. After cloning that gene, they placed it under the control of a promoter sequence from another species that in normal circumstances regulates the production of one of several proteins called antifreeze proteins (because they protect the northern ocean fishes from death due to near-freezing water temperatures). A promoter is a short stretch of DNA, usually located just before the structural gene, that controls its timing and level of activity. Proteins are made when a regulatory protein binds to the promoter and tells it to turn the relevant gene on. The transgenic Atlantic salmon are not huge merely because they have a new promoter sequence. The real change is that the artificially constructed gene is steadily expressed in the fish liver, not just occasionally expressed in the pituitary. Yet, blood levels of the hormone in the transgenic fish are not abnormally high, probably due to the speed with which the hormone is taken up by cells.

After they souped up the salmon GH gene by giving it a very active promoter, the scientists injected the construct (as it is called) into early salmon embryos. Some cells in some of the embryos took up the gene and incorporated it into their genomes, including cells that would eventually create their reproductive systems. When a pair of the genetically engineered fish born after this transgenic manipulation mate, they pass on two copies of the more powerful growth hormone gene to their offspring. A single fish can have thousands of offspring. In nature most do not survive, but in a fish farm their odds are much improved.

The effort to produce and market transgenic salmon and trout is at the leading edge of the "Blue Revolution," a movement to use sea creatures to feed the world. The United Nations has estimated that in the long run, the only way to sustain a world population of 10 billion people is to vastly increase (at least 7-fold the current level) the food yield from the oceans in a manner that does not deplete them. Land-based aquaculture may hold part of the answer, with the special dividend that it does not want to redefine the oceans as vast watery farms.

How will consumers react to transgenic fish? That depends on many factors, including their perception of its safety, the look and taste of the fish, and its cost. There is no rational reason to worry about whether it is safe to eat. The transgenic salmon has no foreign genes and produces no altered proteins. It does make more growth hormone than its smaller cousins, but that protein is quickly metabolized. On repeated assays, the flesh, which looks and feels the same as ocean-grown salmon, has normal levels of growth hormone in it. How does it taste? A/F Protein recently hosted a dinner at The Stone House, one of Canada's premier restaurants, that featured its transgenic salmon and trout. Several of Canada's most highly regarded chefs sat down to dinner with government officials and tried a variety of fish-based dishes. The verdict? The chefs declared AquAdvantage trout and salmon to be absolutely delicious and indistinguishable from the finest ocean-caught fish!

Such testimonials are not likely to satisfy the "Pure Food Campaign," a coalition of advocacy groups directed by perennial biotechnology critic, Jeremy Rifkind. Like similar groups in Europe, it wants any food product that has in any way been genetically altered to be labeled so that the consumer can factor that into his or her decision to purchase. The management of A/F Protein, Inc. completely agrees. Elliot Entis plans to label his products whether he is required to or not because he is so confident that consumers will find them much more desirable than the more expensive ocean-caught fish. He hopes to satisfy both U.S. and Canada regulatory requirements in 2000 and have the fish in stores by 2001. Price-weary consumers may then see a dramatic drop in the cost of salmon, possibly to below that of beef.

Once they learn more about transgenic fish, at least some staunch environmentalists should be converted. These fish will be cultivated in land-based breeding pens, which are much less dangerous to shoreline environments than typical offshore fish-breeding facilities, which generate so much harmful waste effluent (largely from having too much fish feces in too small a space) that they can seriously harm the microflora of the surrounding shallow ocean waters. Furthermore, there should be less need for offshore facilities, creating less demand to use aesthetically attractive shorelines for commercial purposes.

What is the major objection to the AquAdvantage salmon and the dozen other aquatic species that A/F Protein is genetically engineering to be bigger and cheaper? Skeptics point out that in agriculture, which has progressed slowly over thousands of years, humans have painstakingly accumulated much knowledge about plant breeding. But in the thousands of years that humans have fished, they have never bred genetic changes into any organism. They fear that transgenic salmon, trout, shrimp, and other species will inevitably escape into the larger world, and that such an event could lead to an ecological disaster. For example, Rebecca Goldberg of the Environmental Defense Fund worries that the AquAdvantage salmon could outcompete its nontransgenic cousin and in a few generations take his place in the ecosystem. It is impossible to completely allay such concerns.

Despite their potential benefits to consumers and the environment, a great political battle over the approval of products like the transgenic salmon may be looming. The world-wide scare over "mad cow disease" in the United Kingdom, an animal brain disease that has been linked to fewer than 20 human deaths, less than the number of people killed in auto accidents on a typical weekend in the United States, has generated fears that are hard to calm, fears that have been exacerbated by some journalists. En-tis recounts that he foolishly spent an hour educating a British tabloid journalist who was grilling him about safety, only to find that the story headline read, "Gene company denies link with mad trout disease," a disease that does not exist.

Ultimately, the future uses of transgenic fish will be decided politically. In Canada, where both the government and many fisherman are in favor of marketing transgenic fish, the outlook for A/F Protein products is good. In the U.S., and even more so in Europe, governments are more cautious and fishermen are not yet pressing for the product. In the U.S. the major political battleground is currently in Maine. Governor Angus King is boldly defending the state's burgeoning aquaculture industry despite criticism from environmentalists who argue that artificially created salmon that escape into the wild and breed with the few remaining "natural" salmon that spawn in Maine rivers are violating their genetic integrity. Nevertheless, the scientists at A/F Protein are confident. Arnie Sutterlin, who supervises the A/F Protein hatchery in Canada, is fond of reminding folks that virtually no consumer knows that the chicken he eats is a highly manipulated descendant of a jungle fowl in Asia. He is convinced that if the fish is safe, tastes good, and costs less, the world will beat a path to their door.

Hormones as Drugs

The ten-year battle over injecting genetically engineered bovine growth hormone into cows to increase milk production shows how difficult it can be to allay consumer concerns about unfamiliar changes to staple products. Dairy farmers have been striving (with great success) to increase milk production in herds for several hundred years. Long before Darwin offered the intellectual explanation for why what they did worked, farmers routinely selected the cows that were the best milk producers to mother their next generation of calves. A major advance occurred in the 1950s when dairy farmers began to use artificial insemination with semen from prize bulls, but even those production gains pale before the gains with bovine growth hormone.

We have been using hormones as drugs since the discovery of insulin in 1927, but for decades our methods for purifying them were crude and the cost of using them was high. The rise of genetic engineering in the 1970s made it possible to gain access to virtually any human or animal protein in theretofore undreamed-of quantities. The field grew out of the discovery in the late 1960s that many species of bacteria make enzymes that help defend themselves against other bacteria which attack them by chemically cutting alien DNA. The defender's enzyme will cut DNA at a particular recognition site (a short sequence anywhere from four to eight base pairs long) that might appear many times in other organisms, but is not part of its own genome. The discovery of these restriction endonucle-ases is one of the most critical developments in modern biology. As scientists have discovered more and more of them, they have built a huge catalog of specialized DNA cutting tools. By using those tools in combination, they can cut DNA up in different ways to get sequences of different lengths. This ultimately let them develop efficient methods to find and clip out genes they wanted to study and to mass produce them.

The dairy industry quickly guessed that if cows were given higher doses of their own growth hormone they might produce much more milk. Because growth hormone genes from several species had already been isolated, it was straightforward to clone bovine growth hormone, mass produce it in bacteria, and study its effects in cows. The hard part was satisfying the Food and Drug Administration that it was safe to use, a process that took ten years. The FDA approved Posilac, the brand name for genet ically engineered bovine somatotropin (BST), which is another name for growth hormone, on November 5, 1993. Approval meant that a team of neutral expert reviewers had concluded that use of BST to increase milk production would be safe to the cows, to the humans who would consume the milk, and to the environment. Within a year, more than 1,000,000 of the nation's 10,000,000 cows were being injected with BST. By 1997 the number had climbed above 2,000,000. Although there is some debate over exactly how much injection of BST increases milk production per cow, even the most conservative estimates exceed 15%.

Even before Posilac came to market, activists founded the aforementioned Pure Food Campaign to fight it. The initial focus on safety to humans did not generate much public response. BST cannot even be detected in the milk itself, and even if there are minuscule levels, the protein would be almost instantly destroyed in the human gut. Like insulin, it could only be active if injected directly into blood. The much respected former Surgeon General, C. Everret Koop, forcefully asserted that the milk was safe. The impact on cows was not quite so benign. Some animal welfare activists were incensed that cows getting BST were more likely to develop mastitis, an infection of the udders. Mastitis is a common problem in dairy cows, a consequence of the century-long effort to maximize milk output. BST may have contributed in a small way to a longstanding problem, but it did not greatly exacerbate the problem.

The concern that generated the biggest public response was that BST was being used by agribusiness, which could afford it, to put more economic pressure on small dairy farmers who could not necessarily afford the drug. The famous Vermont-based ice cream maker, Ben & Jerry's, even labeled their ice cream containers with the slogan "Save family farms—No BGH." The greatest measure of the success of the consumer activists was in August, 1993, when Congress temporarily banned the sale of the hormone, but that ban was lifted after Monsanto secured FDA approval. Ultimately, the biggest benefit of widespread use of BST may be environmental. If it permits farmers to meet production goals with fewer cows, the nation's dairy herds will release less methane gas into the atmosphere and their manure will pollute fewer streams.

The early use of genetically engineered hormones to treat humans did not trigger nearly the public outcry that the use of BST in cows evoked. This was probably because the hormones were being offered to children for whom no other therapies were available. During the 1960s and 1970s several thousand children with primary growth hormone deficiency who desperately needed human growth hormone if they were to avoid going through life with extreme short stature (a final adult height not much more than that of a 4-year-old) depended on human cadavers for their medicine. The growth hormone, which was always in short supply, was extracted from pituitaries, purified, and injected. Use of human growth hormone from cadavers was banned by the FDA in 1985 when several patients developed a fatal brain disorder (Creutzfeldt-Jakob disease) from a virus that had been transmitted in the extracted pituitary glands.

Serendipitously, in October of that same year, Genentech, among the first and most famous biotechnology companies, received approval from the FDA to market a genetically engineered form of human growth hormone. Cadaver pituitary glands were no longer needed. The new drug, named Protropin, only the second genetically engineered drug to be approved, was manufactured by cloning the gene for human growth hormone and inserting it into a weakened strain of E. coli bacteria. Genentech fermented trillions of these bacteria in huge tanks and then purified the hormone (which is chemically identical to the naturally occurring form) from the broth to make a compound that was safe to inject into children. A few children do suffer from side effects, including diabetes and reduced thyroid function, because their bodies develop an immune response to what they sense to be a foreign protein. Protropin and virtually identical versions made by several competitors are now in wide use to treat severe short stature.

Over the last decade, as more people have heard about human growth hormone, many parents have requested it for children who do not have a growth hormone deficiency, but who appear destined to be among the shortest of adults. A growing number of studies have indicated that the regular injection of hGH in some of these children will modestly increase their final adult height. Requests to use the drug to treat routine short stature raises several ethical issues that may be considered surrogates for a similar debate over the scope of gene therapy in the future. At what height is someone so short that there is good reason to treat him or her? Who decides that question? Should anyone who wants to use hGH as a growth enhancer and who is willing to pay the hefty fee for it have the right to use it? Advances in medicine, especially in gene therapy, will blur the line between interventions to treat an illness and those to enhance the capabilities of a healthy person.

Animal Factories

Compared to their concern over consuming the meat of transgenic animals or the milk of cows that have been given genetically engineered drugs, the public seems much more favorably disposed to using transgenic animals to produce precious medicines, says Jim Geraghty, who should know. Until recently, Geraghty was president of Genzyme Transgenics, a company that is attempting to revolutionize the pharmaceutical industry by using transgenic goats as living factories. No such drug is yet on the market, but soon could be.

Genzyme Transgenics operates a factory of the future: Instead of computer-programmed robots, its tools are several hundred purebred goats imported from New Zealand. Since 1990 Genzyme scientists at a farm in central Massachusetts have been creating transgenic goats that carry in their genomes one of a growing number of human genes. They use these animals as the founders for all their transgenic lines, a process that takes about 18 months. In each case, the scientists use the same basic approach. They start with a cloned ^-casein gene, which produces the most abundant protein in goat's milk, with which they combine the human transgene of interest. They then use extremely fine glass needles to insert this DNA construct into early goat embryos. After microinjecting the gene of interest into the embryos, the scientists transfer them into the wombs of surrogate mothers for a 5-month gestation. After the surrogate mothers give birth, it takes another 8 months for the kids to become mature enough to themselves become pregnant. After the transgenic goat gives birth, it produces milk that expresses the foreign protein. Eighteen months is about the amount of time that companies devote to building brick and mortar fermentation facilities to produce rare proteins. But using a few dozen goats as one's factory is potentially much cheaper. Once the dedicated transgenic goat herd is created, it takes only 30 goats a year to make 100 kilograms of the protein, about as much as a huge factory might yield.

The first protein that Genzyme Transgenics has manufactured in goat factories is antithrombin-III (AT-III), a blood-thinning protein that is of great potential value in preventing blood clots which can cause both heart attacks and strokes. By February of 1994, the molecular biologists had succeeded in developing transgenic goats from which they were harvesting seven grams of antithrombin-III from every liter of goat's milk. The company is now using the same strategy in a dozen or more collaborations with pharmaceutical companies, including the manufacture of a bone growth factor, a monoclonal antibody targeted against colon cancer cells, alkaline phosphatase which is a commonly used test reagent, and several established protein anticancer agents. Genzyme Transgenics thinks it can produce rare proteins at 1% of the cost of traditional cell culture facilities.

Efforts to turn goats into bioreactors for a precious protein usually begin with a study to determine whether it can be produced in mice, the animals with which scientists have the most experience in integrating "transgenes." The first transgenic mice are a kind of pilot plant. If one can harvest the gene product of interest from them, it justifies the decision to scale up to a larger animal that can produce it in large quantities. No human gene seems too large or complex for the system. In 1994 Pharmaceutical Proteins Limited (PPL) of Edinburgh, the commercial sister of the Roslin Institute, the research group that cloned the sheep named Dolly (see Chapter 23) and which pioneered the creation of protein-yielding livestock, announced that in partnership with scientists at Zymogenetics, Inc. (Seattle) it had created a mouse that makes milk loaded with fibrino-gen, a key protein in the blood-clotting cascade. Fibrinogen is composed of six interconnected protein chains that are made by three different genes; one copy of it weighs the same as 340,000 hydrogen atoms. In an even more impressive tour de force, two California biotech companies, Cell Genesys and GenPharm International, created mice that make human monoclonal antibodies, work which could lead to an important new set of weapons to fight cancer. Scientists have even found genes for enzymes called furins that perform the final touches (called posttranslational modification) in protein manufacture, such as adding sugar molecules, and have succeeded in inserting them into the embryos of goats as well. The presence of these genes in the goat will make the proteins in the goat milk of even higher quality.

Since the arrival of Dolly, there have been astounding advances in mammalian cloning. Among them, Genzyme Transgenics recently announced the birth of three goats, each of which is a clone of the same transgenic goat embryo and all of which contain the human gene for antithrombin III. This was accomplished by taking cells from a female goat embryo already known to carry the human Atlll gene and transferring the nucleus of each into a separate enucleated goat egg. The eggs are then transferred to surrogate mother goats that have been hormonally readied for pregnancy. Of 112 cloned embryos placed into 38 surrogates, three clones were born. This may seem low, but it represents a definite advance in the efficiency with which animals can be used as bioreactors (Dolly was the only success in 277 attempts). By cloning a female embryo, the researchers will of course produce only female offspring, all capable of producing milk with the human Atlll. Sandra Nusinoff Lehrman, who is the current CEO of Genzyme Transgenics, estimates that more than $200 million is spent in Europe each year to purchase Atlll.

In March of 2000, PPL Therapeutics, the biotech firm in Scotland that helped clone Dolly, announced that it had produced its first litter of cloned pigs. This is a key step in the effort to create pigs that have a human gene which will insulate their kidneys from rejection if transplanted into humans (Chapter 16).

Such advances are provoking protests, however. Until Dolly, the most famous farm animal in the world was Herman, the first transgenic goat in Europe, created in 1990 by a company in The Netherlands called "Pharm-ing." At a 1997 scientific meeting, George Van Beynum, one of the company's executives, contrasted the generally positive response of the American public to the arrival of such animals with the deep uneasiness in his home country. The Netherlands has a law making it a crime to create transgenic animals unless one has first obtained a permit from the Minister of Agriculture, a permit which, due to political pressure, is almost impossible to obtain. Sustained pressure from animal rights groups in Denmark, Sweden, Germany, and Austria could lead those nations to adopt similar rules, which will make it much harder for their scientists to compete.

In the United States, one development that has enraged some animal rights groups is the practice of awarding patents on transgenic animals. One of the most famous patents in history is for the "Harvard mouse," a transgenic animal created in the laboratory of the renowned geneticist, Philip Leder, that carries in its genome a human gene with mutations that make it highly likely to develop breast cancer. Although the U.S. Patent Office had been issuing patents on inbred strains of plants and animals for decades, the award of the patent to Harvard in 1987 for a mouse engineered to carry a human gene struck some people as a new level of arrogance in the history of our domination of other species. In Europe during the mid-1990s, groups skeptical of genetics nearly won a battle over the language of the proposed European patent directive that could have made it much more difficult to secure the intellectual property rights to new products made by manipulating DNA. This would have surely been a pyrrhic victory, as it would seriously hamper the prospects for biotechnology in those countries.

Mice

Since I have been discussing the contributions of genetically altered animals to human welfare, I would be remiss here not to speak a few words of well-deserved praise on behalf of mice (Mus musculus). They are small, hearty, easy and inexpensive to care for, and in many ways remarkably like people. Scientists have been studying every aspect of their lives, from genes to courtship behavior, for decades. Because we already know them so well, powerful new tools in molecular biology promise to let us know them much better still. When the Human Genome Project, the federally funded effort to sequence all 3,000,000,000 DNA letters that make up the human blueprint, was launched in 1990, the planners knew that it was essential to develop in parallel equivalent efforts to learn the genetic maps and fine structures of other organisms. They chose the mouse. Among higher organisms, the level of detail of the genetic map of the mouse is today second only to that of humans.

Despite the fact that humans and mice have to go back more than 50 million years to find an ancestor that they share in common on the evolutionary tree, they are, comparatively speaking, cousins. Humans and mice have far more similarities than differences in their genes. This is because once nature has worked out an efficient solution to a challenging problem in cell physiology, it tends to reuse it in new organisms. Through the eons only modest changes drift into the DNA, and many of them are neutral in impact. That is, they do not really change the function of the protein for which they code. In two widely divergent species the protein differences may be no greater than say the performance differences between two comparably priced four-door sedans both using the same engine, but manufactured and marketed under different names.

Although we so far know the sequence of only a small fraction of the DNA in the mouse and human genomes, we can say with confidence that coding sequences are identical for about 80% of the base pairs. In most instances, the order in which genes sit on chromosomes is also the same in humans as in mice. Over the years, this has allowed scientists to compare gene locations in the two organisms and conclude that, for example, mouse chromosome 16 has many of the same genes lined up in the same order as does human chromosome 21.

Human and mouse gene mapping are in full swing at hundreds of labs around the world, and new data are poured into publicly accessible (via the World Wide Web) databanks every day. One of the first questions that a scientist now asks when he or she finds a gene of unknown function is whether or not a structurally comparable gene exists in other species. The first stop is often a computerized search of the mouse genome database. Just by finding that the mouse has a comparable gene suggests to the human geneticist that both are involved in some basic biochemical function. Often, the human geneticist may discover that a lot of hard work has been done for him. If a mouse geneticist has figured out what the gene does, it is very likely that in humans the comparable gene has the same duties.

With a powerful tool called site-directed mutagenesis, researchers can now deliberately destroy specific genes in mouse embryos in an effort to develop models of human diseases. These "knock-out" mice are today playing a key role in our pursuit of the causes of disorders ranging from breast cancer to Alzheimer disease. One of the first knock-out mice was designed to mimic Tay-Sachs disease, a fatal brain disorder of childhood caused by mutations in a gene that codes for a protein known as hex-osaminidase A, which normally breaks down waste molecules inside the cell structures known as the lysosomes. In 1994 Richard Proia, a researcher at the National Institute of Diabetes and Digestive and Kidney Diseases at the NIH, crippled the comparable genes in mice embryos. As hoped, the mice grew up to have abnormal lysosomes, which makes them useful models to study the way the waste molecules accumulate in the brain. Oddly enough, however, these mice are relatively healthy. A few other examples of engineered mice include mice that have been created lacking a gene critical to the health of the placenta (they survive because healthy cells are transferred into the embryo at a key point to save a failing placenta), mice that lack a gene for a protein involved in the structure of myelin (which insulates nerves), mice that provide a model for multiple sclerosis, mice that lack one copy of the murine equivalent of the Huntington disease gene (and which have a reduced number of cells in the same parts of their brains as do human patients), and mice that lack the p16 gene, a condition which makes them susceptible to cancer. In one experiment, 9 out of 13 had spontaneously developed cancer by 26 weeks of age. The Alzheimer knock-out mouse was created by deleting the mouse version of a gene called presenilin-1 and inserting a mutated form of the analog human gene that is a cause of a rare hereditary form of the disease into mouse embryos. Scientists at several medical schools are working to develop other mouse mimics of Alzheimer disease. Together these animals permit a level of study undreamed of as recently as the 1980s. So the next time you see a mouse in your basement, think twice before you reach for a weapon. The quirky little guy has some cousins who might be working to save your life!

Please see print version of this book for this figure.

The Florida panther, highly inbred and nearly extinct, is being saved by the introduction of a closely related variety from Texas with which it can breed. (Photo © Larry W. Richardson.)

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