IN THIS ISSUE: Phytoremediation of Volatile Pollutants through Genetic Engineering Genetically Engineered High Lysine Corn Camelina sativa: A Potential Oilseed Crop for Biofuels and Genetically Engineered Products Will Regulation Prevent Animal Biotech From Bringing Home The Bacon? PHYTOREMEDIATION OF VOLATILE POLLUTANTS THROUGH GENETIC ENGINEERING Sharon Lafferty Doty and Stuart E. Strand Improper chemical disposal and spills have resulted in widespread contamination of the environment. Some of these contaminated areas, termed Superfund sites, are polluted to dangerously high levels. In addition, there are over 500,000 contaminated industrial properties in the United States alone that have been abandoned due to the high cost of clean up. Billions of dollars are spent each year in attempts to remediate polluted sites. Engineering methods for the remediation of contaminated sites include excavation, transport, soil washing, extraction, pumping and treating of contaminated water, addition of oxidants, or incineration. Another common clean up method, bioremediation, involves the use of specific microbial strains or communities known to degrade the pollutant. Phytoremediation is the use of plants to clean up contaminated sites (for recent reviews, see 1,2). Phytoremediation is basically a solar driven pollutant extraction system to remove pollutants from water, soil, and air. It is considerably less expensive than the other methods; it is less intrusive and more aesthetically pleasing. By acting as soil stabilizers, plants minimize the amount of contaminated dust that leaves the site and could enter the surrounding neighborhoods. Unlike bioremediation done with microorganisms, phytoremediation is more easily monitored; the condition of the plants can be determined visually; and samples of plant tissue can be easily collected and tested for the presence of the pollutant over time. Phytoremediation is primarily an aerobic process, and its use can avoid the production of intermediates with increased toxicity that is characteristic of some bioremediation methods. Another advantage of phytoremediation over the engineering or bioremediation methods is the possibility of a producing a useful product, such as wood, pulp, or bioenergy, that could offset some of the overall cost of the remediation. Phytoremediation of trichloroethylene and carbon tetrachloride Phytodegradation refers to the process in which plants break down pollutants, either through internal or secreted enzymes. Phytodegradation of chlorinated hydrocarbons and explosives has been studied the most extensively. Trichloroethylene (TCE), one of the most common groundwater pollutants, and carbon tetrachloride (CT) are suspected human carcinogens. Hybrid poplars (Populus trichocarpa × P. deltoides) take up and degrade TCE, producing the same TCE metabolites as mammals3,4, and also take up and degrade CT5,6. In controlled field studies with hybrid poplars, the trees removed over 99% of the added TCE7 or CT6. Transpiration of TCE and CT from leaves and trunks was negligible. In order to determine if poplar cells have an inherent ability to degrade TCE and CT or if microorganisms are responsible for the degradation, studies were conducted with suspensions of pure poplar cell cultures. When these poplar cell cultures were dosed with TCE, the same metabolites that were found in the whole plant studies were also seen4,8,9. Experiments with poplar culture cells and whole plants demonstrated that the primary metabolite, trichloroethanol, is glycosylated as in mammalian systems8. Poplar culture experiments with CT showed that plant metabolism of CT was inhibited by the same chemicals that inhibit CT metabolism by P450 cytochromes in mammalian systems5. Disadvantages of phytoremediation The primary disadvantage of phytoremediation when compared to engineering methods is that it is often too slow or only seasonally effective. Regulatory agencies often require significant progress in remediation to be made in only a few years, making most phytoremediation applications unsuitable. For some pollutants such as TCE and CT, the concentration of the pollutant is not reduced sufficiently to meet regulatory requirements. In some contaminated sites, the pollutants can be at phytotoxic concentrations or recalcitrant to degradation such that plants are not effective. For these reasons, attention recently has focused on ways to enhance the phytoremediation potential of plants using genetic engineering. Genetic engineering of plants for enhanced metabolism of pollutants A direct method for enhancing the effectiveness of phytoremediation is to overexpress in transgenic plants the genes involved in metabolism, uptake, or transport of specific pollutants (recently reviewed in 10-12). This can be readily achieved for many plant species by using Agrobacterium tumefaciens-mediated plant transformation. Since phytoremediation is generally more effective when using large, fast-growing plants, the focus has been on poplar trees. Depending on the hybrid and particular clone, reasonable transformation frequencies can be achieved in poplar13. To increase the phytoremediation potential of the common pollutant TCE, we genetically engineered plants with a mammalian cytochrome P450 enzyme known to metabolize it. The P450 2E1 enzyme controls the rate-limiting step in the metabolism of multiple environmental pollutants, including TCE, carbon tetrachloride, chloroform, benzene, vinyl chloride, and ethylene dibromide. When the cytochrome P450 2E1 gene (hCYP2E1) was overexpressed in tobacco plants, metabolism of TCE was substantially increased14. Furthermore, the transgenic tobacco removed 98% of the ethylene dibromide, compared with 63% removal by the null vector control plants. The P450 2E1 enzyme from rabbit was successfully expressed in hairy root cultures of Atropa belladonna15. These mammalian enzymes functioned well in plants without any need to modify the gene or to include the other enzymes, oxidoreductase and cytochrome b5, known to be required for full function of mammalian P450s. These common enzymes seem to be sufficiently similar in mammals and plants such that the P450s can function with either type. In recently published work, clear enhancement of phytoremediation potential was obtained when the rabbit 2E1 gene was overexpressed in hybrid poplar (P. tremula × P. alba)16. TCE metabolism in two of the transgenic poplar lines was enhanced more than a hundred-fold, with an overall enhancement of over 40-fold in the transgenics compared to the control plants. The transgenic poplar clone with the highest expression of CYP2E1 removed TCE faster than other plant lines, taking up TCE at a rate 53-fold faster than the controls. The CYP2E1 transgenic poplar removed other P450 2E1 substrates, including chloroform, CT, and vinyl chloride from the hydroponic solution, at faster rates than did the control plants. When the transgenic plants were exposed to the volatile form of benzene and TCE, they removed these compounds from the air faster than the control plants. While the control plants barely removed any TCE from the air, the transgenic poplar removed 79% of the TCE during the one-week experiment. Therefore, overexpression of a single enzyme can lead to dramatic improvements in phytoremediation potential of a variety of pollutants from both water and air. By increasing the metabolism of TCE within the plant, lesser amounts of the unaltered compound would be released into the atmosphere via phytovolatilization. Safety concerns Due to the strict regulations governing the release of transgenic trees, it is likely that genetically engineered trees for phytoremediation will be used only on closely monitored sites such as SuperFund sites or military installations where the unintentional spread of the plant material would be unlikely. To prevent transgene flow, the trees would be cut down before they became sexually mature, after several years in the field. Careful selection of the species to be transformed can avoid routes of transgene release by using trees that will not resprout from wind-blown branches. If the transgene did "escape" into native populations, a gene involved in pollutant degradation would be unlikely to confer any selective advantage or negative environmental impact. Conclusions There are numerous reports in the literature supporting phytoremediation as an effective method in treating hazardous sites. Yet it is not used as widely as it could be. In the last several years, significant progress has been made to increase the efficiency of phytoremediation. Using genetic engineering, substantial increases in removal rates of hazardous pollutants, including nitroaromatics, solvents, and metals, has been achieved. With the increased effectiveness of phytoremediation, this "green technology" may be used to decrease the expense of clean-up and lead to an increased likelihood that sites will be restored rather than abandoned. Reference List 1. Suresh B. and Ravishankar GA (2004) Crit Rev Biotechnol24, 97-124 2. Pilon-Smits EAH and Freeman JL (2006) Front Ecol Environ4, 203-210 3. Gordon MP et al. (1998) Environ Health Perspect106, 1001-1004 4. Newman L et al. (1997) Environmental Science and Technology31, 1062-1067 5. Wang X, Gordon MP, and Strand SE (2002) Biodegradation13, 297-305 6. Wang X et al. (2004) Environ Sci Technol38, 5744-5749 7. Newman L et al. (1999) Environ. Science and Technol33, 2257-2265 8. Shang QT et al. (2001) Phytochemistry58, 1055-1065 9. Shang QT and Gordon MP (2002) Chemosphere47, 957-962 10. StompAM, et al. (1994) Annals of the New York Academy of Sciences721, 481-491 11. Rugh CL (2004) Trends Biotechnol22, 496-498 12. Cherian S and Oliveira MM (2005) EnvironSciTechnol39, 9377-9390 13. Han K-H et al. (2000) Plant Cell Reports19, 315-320 14. Doty S L et al. (2000) Proc Natl Acad Sci USA97, 6287-6291 15. Banerjee S et al. (2002) Biotechnol Bioengineering77, 462-466 16. Doty SL et al. (2007) Proc Natl Acad Sci USA104, 16816-16821 Sharon Lafferty Doty College of Forest Resources University of Washington, Seattle, WA sldoty@u.washington.edu Stuart E. Strand College of Forest Resources University of Washington, Seattle WA sstrand@washington.edu GENETICALLY ENGINEERED HIGH LYSINE CORN Shihshieh Huang, Alessandra Frizzi and Thomas Malvar High yield, but a poor protein source Thanks to the development of hybrid varieties in the1950s, corn has become the most productive major crop species in the United States. On a per acreage basis, corn yield exceeds that of the other two major crops, wheat and soybean, by 2- to 3-fold. However, the nutritional quality of corn protein in these high yielding hybrids remains relatively poor due to its deficiency in essential amino acids, such as lysine. The focus of corn breeding on yield, which has resulted in a shift in grain composition from protein to starch1, has compounded this nutritional deficiency. As a major food and feed staple crop, corn is a poor protein source in both quality and quantity. Advancements in agricultural biotechnology may help improve the nutritional quality of corn protein, starting with lysine enhancement. Strategies for lysine genetic engineering in corn Corn is mainly used as animal feed in developed countries. The most limiting amino acid in corn-based feed is lysine, and this insufficiency is overcome by supplementing corn meal with soybean meal or crystalline lysine produced via fermentation. To broaden the use of corn meal, several transgenic approaches have been investigated — at least two examples of genetically engineered high lysine corn have been successfully demonstrated in field-grown inbred crops. Natural maize opaque mutants have a better dietary protein profile due to low levels of the nutritionally poor corn protein known as α-zein. Therefore, RNAi has been used to specifically suppress α-zein producti on in transgenic corn, resulting in a doubling of the lysine content of corn grain from 2400 ppm to 4800 ppm2. α-zeins comprise roughly 40% of the total kernel protein, but contain almost no lysine. By reducing α-zeins, other lysine-containing kernel proteins were comparatively increased, raising the lysine content in corn protein from 2.8% to 5.4%. However, like the original opaque mutants, the soft and chalky kernel phenotype displayed by these transgenic lines remains a deterrent to commercialization. Alternatively, the plant lysine metabolic pathway provides possible enzyme targets for genetic engineering to increase free lysine content in corn grain. As shown in Fig. 1a, lysine, along with methionine, threonine, and isoleucine, is derived from aspartate; dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step of lysine biosynthesis. A bifunctional enzyme, lysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH), is responsible for lysine catabolism3,4. The free lysine level in plant cells is thought to be regulated by lysine feedback inhibition of DHDPS and feed-forward activation of LKR/SDH. Indeed, the expression of a lysine feedback-insensitive DHDPS from Corynebacterium glutamicum, CordapA, as well as the suppression of LKR/SDH have resulted in transgenic corn with higher levels of free lysine5,6.

A bifunctional transgene for high lysine corn To further enhance the accumulation of free lysine in corn, we recently developed transgenic corn lines that combine CordapA expression and LKR/SDH suppression7, by using a novel bifunctional transgene cassette (Fig. 1b). An inverted repeat sequence corresponding to partial LKR/SDH cDNA was inserted into the intron of an expression cassette containing CordapA as the coding region. In principle, the expression of this transgene should generate an intron-derived, double-stranded RNA against LKR/SDH and an mRNA encoding CordapA. Western blot analysis of R1 seeds confirmed that the detection of CordapA was accompanied by the reduction of LKR/SDH in transgenic kernels (Fig. 2). Further detailed molecular analyses of these transgenic plants are presented in the original research article7.

As summarized in Table 1, expression of CordapA in the endosperm does not elevate free lysine in mature corn kernels (however, free lysine accumulation was achieved when CordapA was expressed in the embryo5). The suppression of LKR/SDH in the endosperm tissue increases free lysine to 1324 ppm (~30 fold increase). The combination of CordapA expression and LKR/SDH suppression in a single transgene produces over 4000 ppm free lysine (~100 fold increase), the highest ever reported in corn kernels.

Intron-embedded double-stranded RNA (dsRNA) The intron-embedded, silencing cassette design described above reduces the number of individual transgene expression cassettes required to genetically engineer the corn lysine metabolic pathway. We also demonstrated that this design can be used to effectively inhibit the expression of Luciferase and GUS, without compromising the expression of a downstream marker gene7. Plant metabolic engineering may often employ multiple transgene cassettes, each requiring its own genetic regulatory elements such as promoters and terminators. This can be challenging due to the limited availability of suitable regulatory elements and the desire to coordinately express multiple transgenes. Incorporating gene suppression elements, such as double-stranded RNA (dsRNA), short hairpin RNA (shRNA), or engineered miRNA, into an intron can conserve regulatory elements while allowing temporal and spatial synchrony of gene suppression and expression when such a strategy is warranted. What's next? Based on the progress made in engineering high lysine corn, it is possible that one day the lysine supplementation of corn meal will no longer be necessary. Engineered corn with increases in other limited essential amino acids such as tryptophan, methionine, and threonine will likely follow. The quantity and digestibility of corn protein in corn grain may also be improved through plant biotechnology. Conventional breeding over the past several decades has made corn one of the most abundant sources for feed and food in the world. Through genetic engineering, the hope is that corn would someday have much greater nutritional value as well. References 1. Scott MP, Edwards JW, Bell CP, Schussler JR and Smith JS (2006) Grain composition and amino acid content in maize cultivars representing 80 years of commercial maize varieties. Maydica51, 417-423 2. Huang S, Frizzi A, Florida CA, Kruger DE and Luethy MH (2006) High lysine and high tryptophan transgenic maize resulting from the reduction of both 19- and 22-kDα-zeins. Plant Mol Biol61, 525-535 3. Azevedo RA, Lancien M and Lea PJ (2006) The aspartic acid metabolic pathway, an exciting and essential pathway in plants.Amino Acids30, 143-162 4. Stepansky A, Less H, Angelovici R, Aharon R, Zhu X and Galili G (2006) Lysine catabolism, an effective versatile regulator of lysine level in plants. Amino Acids30, 121-125 5. Huang S, Kruger DE, Frizzi A, D'Ordine RL, Florida CA, Adams WR, Brown WE and Luethy MH (2005) High-lysine corn produced by the combination of enhanced lysine biosynthesis and reduced zein accumulation.Plant Biotechnol J3, 555-569 6. Houmard NM, Mainville JL, Bonin CP, Huang S, Luethy MH and Malvar TM (2007) High-lysine corn generated by endosperm-specific suppression of lysine catabolism using RNAi. Plant Biotechnol J5, 605-614 7. Frizzi A, Huang S, Gilbertson LA, Armstrong TA, Luethy MH and Malvar TM (2008) Modifying lysine biosynthesis and catabolism in corn with a single bifunctional expression/silencing transgene cassette.Plant Biotechnol J6, 13-21   Shihshieh Huang Calgene Campus Monsanto Company Davis, CA Shihshieh.huang@monsanto.com CAMELINA SATIVA: A POTENTIAL OILSEED CROP FOR BIOFUELS AND GENETICALLY ENGINEERED PRODUCTS Chaofu Lu Camelina [Camelina sativa (L.) Crtz.] is an ancient crop belonging to the Brassicaceae family. It has been cultivated for oil production since prehistoric times, and it was extensively grown in Europe in the 19th century. However, Camelina fell into disfavor when more productive crops such as wheat and rapeseed began to be produced, and camelina production gradually declined and almost vanished after World War II. It became a common weed in Europe known as false flax (contaminating flax fields) and by its Roman name, Gold-of-Pleasure. The recent interests in camelina are inspired by its unique oil composition. About 90% of fatty acids in camelina oil is unsaturated. The nutritionally essential polyunsaturated fatty acids (linoleic acid, 18:2n-6 and α-linolenic acid, 18:3n-3) constitute over 50% of total fatty acids, and linolenic acid is the most predominant fatty acid (35-40%). Therefore camelina oil has great potential as an excellent source of omega-3 fatty acids1, which have been recommended in the diet to achieve essentiality and cardiovascular benefits. Non-food uses of camelina oil have also been exploited for the production of soaps, varnishes, and cosmetics2, 3. Because camelina has lower fertilizer and pesticide requirements, the production cost is substantially lower than many other oil crops such as rapeseed, corn, and soybean; therefore camelina is an attractive potential crop for biodiesel and many other industrial applications2. Currently, the lack of a clear utilization pattern of camelina oil limits its uses and large-scale production despite its adaptation to a wide range of climates2. Camelina oil contains about 15% eicosenoic acid (20:1), which is unique among many other vegetable oils. The usefulness or disadvantage of eicosenoic acid is not clear; however, it may present a hurdle to obtaining food approval1. The high percentage of polyunsaturated fatty acids makes camelina oil more susceptible to oxidation and thus is undesirable for fuel and other industrial applications. Therefore it is necessary to modify camelina oils to find a role for this potential crop in the world oilseed market. Since 2002, researchers at Montana State University have initiated a research program to evaluate and scale up production, improve camelina characteristics, and explore the utilization of camelina oil and meal. In this report I will present an overview on our recent efforts to improve fatty acid compositions of camelina oils, and to develop camelina as a potential platform for production of biotechnological products through genetic engineering. Screening camelina mutants for desired fatty acid composition We conducted an experiment to screen camelina mutants that contain desired fatty acid compositions. To induce mutations, camelina seeds were treated with ethane methyl sulfonate (EMS). The seeds from individual M2 plants were subject to analyses using high-throughput gas chromatography. This method, as described previously4, has a capacity to analyze about 600 samples a day. We screened over 50,000 M2 lines and identified a number of mutant lines that contain altered contents of different fatty acids, e.g., oleic (C18:1), linoleic (C18:2), linolenic (C18:3), and eicosenoic (C20:1) acids. A mutant with significantly increased oleic acid content is given as an example in Fig. 1.

Oleic acid is the most predominant fatty acid resulting from fatty acid synthesis in plant cells. Much of the oleic acid will be desaturated or otherwise modified during storage oil biosynthesis. In camelina, about 80% of C18:1 will be desatured to C18:2 and C18:3, or elongated to C20:1 and C22:1. Typically, camelina oils contain over 50% polyunsaturated fatty acids, which may render oil instability during storage and applications at elevated temperatures. Therefore high-oleic camelina oil is desirable for food and non-food uses. An efficient method of camelina genetic transformation Camelina is a low-cost oilseed crop that is not at present a major crop for food oil production; therefore it has great potential to become as a biotechnological platform for genetically engineered products. There have been limited research activities on camelina biotechnology. However, like many cruciferous oil producing plants such as Arabidopsis thaliana and Brassica napus, camelina is amenable to transformation. We have recently developed a simple, in planta transformation method for Camelina sativa5. Instead of a lengthy and laborious tissue-culture based method (United States Patent No. 20040031076), we generated transgenic camelina plants by vacuum-infiltration treatments of flower buds using Agrobacterium. Greater than 1% transformation efficiency can be achieved. To demonstrate that camelina can be effectively used to produce genetically engineered products, we transformed camelina seeds with a castor fatty acid hydroxylase (FAH12) gene4 (Fig. 2a). We used a fluorescent protein selection marker DsRed (Clontech, Mountain View, CA, USA) driven by the constitutive cassava vein mosaic virus promoter to facilitate screening transgenic seeds. The transgenic fluorescent seeds (Fig. 2b) could be visually detected using a pair of red-lens sunglasses by illuminating a sea of seeds with a green LED flashlight. The transgenic events were verified by PCR analyses using primers for the DsRed and FAH12 genes (Fig. 2c). Fatty acid methyl ester analyses by gas chromatography (Fig. 2d) indicated that all red seeds analyzed accumulated novel fatty acids, which had been previously identified in transgenic Arabidopsis as ricinoleic acid (18:1OH), the major component of castor oil, and three other hydroxy fatty acids: densipolic acid (18:2OH); lesquerolic acid (20:1OH); and auricolic acid (20:2OH)6. This result clearly confirmed that the red fluorescent seeds were successfully transformed and expressed the castor FAH12 gene.

Conclusion Camelina is an under-exploited crop species of great potential economical importance. We believe that camelina oils with improved fatty acid compositions will be useful for a variety of food and non-food uses. Our transformation system would provide a useful tool to rapidly improve many agronomic charactersistics, as well as a model system for biological and biotechnological studies. In this report, we demonstrated that novel hydroxy fatty acids were produced in camelina oils by seed-specific expression of a castor fatty acid hydroxylase. The low-cost oilseed crop, Camelina sativa, has great utility as an economical platform for a plethora of genetically engineered industrial and pharmaceutical products. References 1. Leonard C. (1998) Camelina oil: a-linolenic source. INFORM9, 830-838 2. Putnam DH, Budin JT, Field LA, and Breene WM. (1993) Camelina: A promising low-input oilseed. InNew Crops, J.E. Simon, Editor. Wiley: New York 3. Zubr J. (1997) Oil-seed crop: Camelina sativa. Ind Crop Prod6, 113-119 4. Lu C, Fulda M, Wallis J, and Browse J. (2006) A high-throughput screen for genes from castor that boost hydroxy fatty acid accumulation in seed oils of transgenic Arabidopsis. Plant J45, 847-856 5. Lu C and Kang J. (2007) Generation of transgenic plants of a potential oilseed crop Camelina sativa by Agrobacterium-mediated transformation. Plant Cell Rep, in press 6. Broun P and Somerville C. (1997) Accumulation of ricinoleic, lesquerolic, and densipolic acids in seeds of transgenic Arabidopsisplants that express a fatty acyl hydroxylase cDNA from castor bean.Plant Physiol113, 933-942 Chaofu Lu, Jinling Kang, David Sands, Alice Pilgeram Department of Plant Sciences and plant Pathology Montana State University, Bozeman, MT 59717-3150 USA clu@montana.edu WILL REGULATION PREVENT ANIMAL BIOTECH FROM BRINGING HOME THE BACON? Henry I. Miller, M.D. Biotechnology is everywhere these days, from the production of pest-resistant crops to microorganisms that make biofuels to new drugs and vaccines. It's even being used to produce animals with novel and valuable traits, but these applications in particular are suffering from inconsistent, uncertain regulation. After 20 years, the FDA has not yet published a policy statement, but a senior official in its Center for Veterinary Medicine recently gave a strong hint of the agency's preferred approach. She said that every new genetic construction in an animal that employs recombinant DNA, or gene-splicing, technology would require approval for use in the food supply, and that the applicable procedures and regulations would be the same as for drugs used to treat animal diseases. But the introduction of a gene is not the same as the administration of a drug. Moreover, the FDA's approach represents a major shift in FDA's regulation of biotechnology that will be hugely expensive to animal breeders and detrimental to consumers. John J. Cohrssen, who worked on FDA reform during the 1990's as majority counsel of the House Commerce Committee, characterized the FDA's new approach as "complex, arbitrary and dilatory." Up till now, the FDA has not regulated farm animals or, for that matter, animals used for what might be termed "medical purposes." For example, if German shepherds or golden retrievers were bred to enhance traits that made them better seeing-eye or companion dogs, the FDA would not regulate them under its medical device or veterinary drug regulations. Nor would a leaner line of pigs be regulated differently from others under the FDA's food regulations, unless some safety issue was raised. Even for transgenic animals used in research, the FDA has not asserted jurisdiction over the hundreds of rodent lines that are available (http://www.criver.com/research_models_and_ services/transgenic_services/tgresearchmodels.htm). The only transgenic animal currently marketed to the public at large is a small, tropical, ornamental (aquarium) zebrafish that glows because of the insertion and expression of a gene that synthesizes a beautifully colored fluorescent protein (http://www.glofish.com). The fluorescent protein genes were obtained from another marine organism, the sea anemone. The FDA opted not to regulate them, under this rationale: "Because tropical aquarium fish are not used for food purposes, they pose no threat to the food supply. There is no evidence that these genetically engineered zebra danio fish pose any more threat to the environment than their unmodified counterparts which have long been widely sold in the United States. In the absence of a clear risk to the public health, the FDA finds no reason to regulate these particular fish." That statement from the FDA would seem to weaken the argument for treating transgenic animals as though they were being treated with a "new drug." (It is noteworthy that in spite of the fact that the fluorescent fish are not eaten and would not survive outside an aquarium, they have been banned by regulators in California.) The most apposite models for gene-spliced or "transgenic" animals are the FDA's oversight of traditional foods and food additives; and the production of livestock clones, or identical twins, which regulators decided last year were safe to eat. A company called Aqua Bounty Technologies has been trying for about a decade to get FDA approval to market an Atlantic salmon that contains a newly introduced Chinook salmon growth hormone gene engineered to keep it turned on all year round (instead of during only the warmer months, as in nature). This cuts the time to marketable adult weight from 30 months to 18. The extra gene confers no detectable differences in the salmon's appearance, taste, or nutritional value; it just grows faster. In spite of sufficient evidence that the fish is safe to eat and does not differ nutritionally from other Atlantic salmon, the FDA has kept the company treading water for almost a decade. There are numerous other applications in various stages of R&D, including transgenic livestock with leaner muscle mass, enhanced resistance to disease, or improved use of dietary phosphorous to lessen the environmental impacts of animal manure. But if regulators don't make appropriate regulatory decisions soon, the entire sector could virtually disappear. One problem plaguing the FDA's Center for Veterinary Medicine is that the "new drug" paradigm doesn't fit transgenic animals well. A better model is the way that another FDA component, the Center for Food Safety and Nutrition, regulates other foods. The law places the burden of ensuring the safety of foods and food ingredients on those who produce them. It prohibits the adulteration (contamination) or misbranding (mislabeling) of food, but the agency does not inspect or evaluate food prior to its sale in shops, supermarkets, or restaurants. Rather, federal oversight relies on market surveillance, or post-marketing regulation, and the FDA takes action only if there is an apparent problem. This approach has worked quite well over many years. The law does require a pre-marketing review for certain food-related products. These include most food additives — a class of ingredients that includes preservatives, emulsifiers, spices, sweeteners, and natural and synthetic flavors or colors, among others. In general, a food additive must be pre-approved if it becomes a component of or otherwise affects the characteristics of a food and it is "not generally recognized as safe (GRAS) by qualified experts for its intended use." GRAS is an important concept: Before a new food additive is marketed, it is the responsibility of the producer to determine whether or not the substance is GRAS. The agency routinely reviews food additive applications for safety only when the substance in question has been determined not to be GRAS by the producer. If the producer determines that a substance is GRAS, only a notification of that decision to the FDA is necessary (which is then subject to agency review). The FDA's existing approach to biotechnology and to foods in general could be adapted easily to transgenic animals. Traditionally, in a logical application of transitivity, the combination of two GRAS substances is still GRAS. Similarly, because adding a GRAS gene to a GRAS organism is likely to yield a GRAS outcome, an FDA pre-marketing review would not be necessary for genetic constructions like the fast-growing salmon. But instead the FDA intends to treat every new animal as though it contains a "new drug," the evaluation of which can take many years even if there is minimal likelihood of harm. The GRAS/food additive concept is relevant to "transgenic" animals because of the nature of the techniques. "Transgenic" animals usually are created by injecting the desired gene — which may be intended to confer an advantage in husbandry or nutrition, for example — into a single-cell embryo, or by inserting the gene into a skin cell and creating an embryo by a process called cloning. In either case, the embryo that now contains the foreign gene is then implanted into the uterus of a surrogate mother. If the foreign gene is incorporated into the DNA of the offspring, then like other genes it is passed on to succeeding generations, and the product of the gene (usually a protein) can be considered either GRAS or a food additive, depending on its function and other factors. These transgenic animals subsequently are propagated in conventional breeding programs. The FDA's approach to "novel" foods, published in 1992, is compatible with the GRAS/food additive paradigm. It emphasizes that the Center for Food Safety and Nutrition does not impose discriminatory regulation based on the use of one technique or another, but that greater scrutiny is applied only when certain safety issues are raised. These include the presence of a completely new substance in the food supply, changes in a macronutrient, increase in a natural toxicant, or the presence of an allergen where a consumer would not expect it. Officials at the FDA's Center for Veterinary Medicine would likely remonstrate that a newly introduced gene expressed in an animal is similar to the injection of a new drug, that the genetic modification mediates the introduction of the substance synthesized under the direction of the new gene — a hormone or vitamin, for example. However, this theory ignores that neither the FDA nor any other government agency routinely conducts pre-market review of new genetic constructions that occur naturally. If animal biotech companies are to bring home the bacon, the FDA will need to establish a sound and consistent regulatory policy soon. Henry I. Miller, a physician and molecular biologist, is a fellow at Stanford University's Hoover Institution and at the Competitiveness Enterprise Institute. He headed the FDA's Office of Biotechnology from 1989 to 1993 and is the author, most recently, of "The Frankenfood Myth." Henry I. Miller miller@hoover.stanford.edu ISB News Report 1900 Kraft Drive #103 Corporate Research Center Virginia Tech Blacksburg, VA 24061 The material in this News Report is compiled by NBIAP's Information Systems for Biotechnology, a joint project of USDA/CSREES and the Virginia Polytechnic Institute and State University. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture, or Virginia Tech. The News Report may be freely photocopied or otherwise distributed without charge. ISB welcomes your comments and encourages article submissions. If you have a suitable article relevant to our coverage of the agricultural and environmental applications of genetic engineering, please e-mail it to the Editor for consideration. Ruth Irwin, Editor (rirwin@vt.edu) To have the News Report automatically e-mailed to you, send an e-mail message to isb@vt.edu with your request. Connect to http://www.isb.vt.edu for internet access to ISB News Reports, textfiles, and databases. Information Systems for Biotechnology, Virginia Tech, 1900 Kraft Drive, Suite 103, Blacksburg, VA 24061, tel: 540-231-3747, fax: 540-231-4434, e-mail: isb@vt.edu