Inhibitors of all four dominant classes of protease (serine, cysteine, aspartic, and metallo proteases) occur in plants. They often accumulate in tissues in response to wounding or herbivory and are an important element of natural plant defence strategies³. Although the exact mode of action of the protease inhibitors (PIs) is not fully understood, their ingestion by insects can result in reduced growth and/or survival. In general, ingestion of serine PIs affects lepidopteran species, whilst cysteine PIs, also referred to as cystatins, affect members of several Coleopteran families, as well as some Homoptera. The expression of PIs in GM plants as an insect defence has been widely investigated⁴ and there is evidence that PIs represent a broad-based resistance strategy for control of plant-parasitic nematodes⁵. Given this broad spectrum of activity of PI's, it is important to develop a framework for assessing the impact of PI-expressing GM plants on non-target organisms. We developed a sequential approach to address this need. In our paper the approach is described using GM nematode-resistant potato plants that express a cystatin, as an example.

Methodology and Results
The sequential approach described in our paper consists of a novel selection phase followed by an assessment phase that is based on testing schemes used to evaluate the effect of pesticides on beneficial insects. The selection phase, which consists of two tiers, is crucial, since it is not feasible to test all potential non-target organisms associated with a particular GM crop. In the first tier, field surveys are used to provide a list of potential non-target invertebrates. In the next tier, histochemical assays are used to identify a subset of potential non-targets that have digestive protease activity in the same class as that targeted by the transgenic PI. In the assays, cryosections of the invertebrates are prepared and incubated with a specific substrate. The non-target insects that consistently show a positive response in the assay are selected for further study in the assessment phase of the approach. This consists of three tiers: `worst-case scenario' laboratory studies, extended laboratory or small-scale field studies, and large scale field trials, which include currently used pest management options.

In our example, the initial field surveys were carried out at 12 sites and included a wide range of potato varieties, crop rotations, and soil types. At seven of the sites, the potatoes were grown conventionally and received synthetic pesticides. The remaining five sites were organic farms without such chemical use. Above-ground plant tissues were visually examined for invertebrates at regular intervals throughout the growing season. Members of five insect families were commonly recorded on the potato crops during the surveys. Aphids were the most prevalent and abundant herbivores during the sampling period and two species, Myzus persicae and Macrosiphum euphorbiae, predominated in the samples. Collem-bola were the next most abundant group whilst Cicadellids (leafhoppers) were prevalent towards the end of the sampling period.

Cryosections of representative species of each of the five families were incubated with a fluorgenic substrate that is cleaved by cysteine proteases. Sites of protease activity in cryosections can be identified because yellow fluorescent crystals, produced by cleavage of the substrate, are deposited on the slide. The subset of non-target organisms that produced a positive response with the assay included the aphid, M. euphorbiae and the leafhopper, Eupteryx aurata. In both species, fluorescent crystals were localized in regions of the cryosections corresponding to the digestive tract of the insects. The presence of cysteine protease activity was confirmed by incubation of cryosections with the substrate and a cystatin. In both species, the level of protease activity was significantly reduced in the presence of the cystatin. As a result, these insects were selected for further study in the assessment phase of the approach. Results for E. aurata are presented in our paper.

In `worst-case scenario' studies, neither the rate of growth nor the survival of E. aurata nymphs were adversely affected by consumption of detached leaves of cystatin-expressing plants. A similar lack of effect was recorded in the next tier of the approach, which involved whole plants grown either under controlled conditions or planted in small-scale field trials. Nymphal survival, rate of development, and the size of emerging adults did not differ between the GM and control lines. There was also no significant variation in the abundance of nymphs on control or GM plants during two years of small-scale field trials. Due to restrictions stipulated in our licence to grow the GM nematode-resistant plants, we were not able to complete the final tier of the assessment phase, large scale-field trials. However, results from the other tiers of the assessment phase suggested that the nematode resistant GM potatoes were not hazardous to a non-target insect shown to have digestive cysteine protease activity.

Discussion
The framework that we have described incorporates several tiers—the techniques used in the various tiers are not in themselves novel. For example, histochemical and biochemical assays have been used to determine sensitivity of insect proteases to plant derived PIs and `worst-case' scenario studies have been used to assess the effect of ingestion of PIs on insect survival. However, results from studies using the different techniques are seldom, if ever, consolidated into a completed risk assessment as is proposed in our approach. The results for E. aurata and for another non-target insect, the aphid M. persicae<sup>6</sup>, highlight the importance of adopting a multi-phase approach. Cysteine protease activity was eliminated in cryosections of E. aurata nymphs when the sections were incubated with a cysteine PI; however, ingestion of cystatin-containing plant tissue did not affect nymphal growth or survival. Similarly, the growth and survival of M. persicae were adversely affected in `worst-case scenario' laboratory studies in which cystatins were incorporated into artificial diets. However, these effects were not reproduced during the next tier of studies using transgenic plants, under both controlled environment and field conditions⁶. Therefore, reliance on single tier experiments, particularly those in which the actual exposure to PIs in GM plants is not considered, could result in an overestimation of the real impact of the plants on non-target species.

The lack of effect of GM nematode-resistant plants on non-target insects may reflect differences in the ability of insects and nematodes to circumvent the adverse effects of PIs. Plant defenses based on PIs tend to be less evident in roots than aerial tissues. Therefore, the feedback loops that leaf feeding insects use to compensate for PIs in their diet, such as the production PI-insensitive enzymes, degradation of PIs, or hyperproduction of proteases, may be less well developed in plant parasitic nematodes, which feed from plants roots. In our study with GM potato, expression in the root system may have been sufficient to confer resistance to the potato cyst nematodes, G. pallida and G. rostochiensis, whilst levels taken up from the mesophyll cells or phloem were insufficient to adversely affect E. aurata and M. persicae, respectively.

The sequential approach described in our paper has been used to evaluate the direct effect of GM nematode-resistant potato plants on non-target herbivorous insects. However, the framework is also applicable to cystatin-expressing plants developed for insect pest management. Similarly, the use of substrates with different specificities in the histochemical assays of the selection phase would allow the approach to be used with inhibitors of other classes of protease, e.g., serine proteases. In its present form the approach does not identify indirect effects on non-target herbivores, such as changes in natural enemy populations, nor does it include herbivores feeding on non-crop plants, such as those that have been shown to be exposed to GM pollen in GM maize crops¹. Nevertheless, we believe the sequential approach has a role in the evaluation of the risks to non-target organisms posed by GM PI-expressing plants.

1. Sears MK et al. (2001) Impact of Bt corn pollen on Monarch butterfly populations: a risk assessment. Proceedings national Academy Sciences USA 98(21): 11937-11942.

2. Cowgill SE & HJ Atkinson. (2003) A sequential approach to risk assessment of transgenic plants expressing protease inhibitors: effects on nontarget herbivorous insects. Transgenic Research (12): 439-449.

3. Ryan CA. (1990) Proteinase inhibitors in plants: Genes for improving defenses against insects and pathogens. Annual Review of Phytopathology (28): 425-449.

4. Jongsma MA & C Bolter. (1997) The adaptation of insects to plant protease inhibitors. Journal of Insect Physiology 43(10): 885-895.

5. Atkinson HJ et al. (1998) Engineering resistance to plant parasitic nematodes. In: The Physiology and Biochemistry of Free-Living and Plant Parasitic Nematodes, eds. Perry RN, & Wright DJ. CABI: Wallingford, UK.

6. Cowgill SE et al. (2002) Transgenic potatoes with enhanced levels of nematode resistance do not have altered susceptibility to nontarget aphids. Molecular Ecology (11): 821-827.

Plants are constantly challenged by changes in environmental conditions. The increasing need for food and consumer preferences necessitates that crop plants are grown in regions where they are not naturally adapted, leading to stress. Reclaiming lands from the sea, seepage of saline water, and excess irrigation lead to increased salinity that plants have to tolerate. Excessive rainfall, flash flooding, or prolonged flooding causes hypoxia and anoxia. A paucity of water due to infrequent rain and inadequate irrigation leads to drought stress. Most of these stresses produce oxidative stress in the plant cells.

Man's activity also produces xenobiotic pollutants such as factory wastes, aerosols, refrigerants, aviation and motor fuel exhaust gases, as well as biotic pollutants such as sewage, which constantly challenge plants. Plant response to stress is complex, as stress may occur at different stages of plant development or plants may experience more than one stress at a time¹. Stress in all its forms has negative effects on plant development and productivity. Plants respond to salinity by reducing leaf growth through inhibition of cell division and expansion. The decrease in the osmotic potential of root cells causes inhibition of water uptake and dehydration of the plants, leading to the death of tissues, organs, and eventually the whole plant. Chilling stress stunts plant growth, brings about cellular autolysis and senescence, and has detrimental effects on flower induction, pollen production, and germination. Chilling and desiccation damage cell membranes while oxidative stress targets membranes, proteins, and DNA.

A serious challenge today is to sustain and improve crop yields to feed the growing world population. Rice is a staple food in much of the world and hence increasing its production is of special interest. Though rice production has increased over the years, it will need to increase another 60% by 2025 to feed the growing population². Traditional breeding strategies have been used to exploit natural genetic variation in improving crop varieties, but until now, very few plants showing enhanced tolerance to stresses and better yields have actually made it to the fields. Thus, genetically engineering plants to increase stress tolerance is a desirable alternative to breeding.

Several genes have been reported to be up- or down-regulated in response to different stresses. These genes might generate products either directly involved in protection against environmental stress or that play a role in stress regulation. The first category would constitute genes coding for osmoprotectants, scavengers of reactive oxygen species, or stress proteins such as COR or LEA with an undefined mechanism of action. In the latter category would be genes that code for regulatory proteins such as transcription factors or components of signal cascades. These proteins would regulate the expression of a set of genes involved in stress. Both categories of genes have been shown to impart tolerance when overexpressed in plants. However, a desirable candidate gene of possible use in crop improvement would likely be a regulatory gene since it would have potential to play a broader role in stress tolerance, imparting tolerance to several stresses.

The OSISAP1 gene was cloned via the differential screening of an indica rice cDNA library in an attempt to identify genes that show organ-specific and/or stress inducible expression³. OSISAP1 was expressed at a higher level in the root and the prepollination stage spikelet as compared to shoot. Further, expression analysis of OSISAP1 revealed that the gene is expressed in response to several abiotic stresses like cold, salt, drought, submergence, mechanical wounding, and heavy metals.

OSISAP1 codes for a zinc-finger protein, which shows homology in the AN1-type zinc-finger region to the human and mouse PRK-1-associated protein AWP1, Phaseolus vulgaris pathogenesis-related protein PVPR3, human and mouse zinc-finger protein ZNF216, Xenopus laevis ubiquitin-like fusion protein XLULFP, and the ascidian posterior end mark protein PEM6. OSISAP1 also shows homology in the A20-like zinc-finger region to AWP1, hZNF216, and mZNF216. A part of the encoded protein also shows homology to human transcription factor NF-kB p65 subunit.

The gene was overexpressed in tobacco under the control of a constitutive CaMV35S promoter to understand its function, and especially to determine whether the gene has a role to play in stress response. Transgenic lines were analyzed for cold, dehydration, and salt stress tolerance in the T1 generation. Germination, fresh weight gain, and bleaching were used as main parameters for comparing the response of nontransgenic and transgenic lines to different abiotic stresses³.

Fresh weight of nontransgenic and transgenic seedlings was measured after 15 days of a recovery period following 15 days of cold stress. Nontransgenic seedlings could gain only 35% fresh weight in comparison to 67—92% fresh weight gained by stressed transgenics when compared to unstressed seedlings. Morphologically, transgenics show better recovery since the third or fourth leaf had already emerged, whereas in the nontransgenic seedlings only the first two leaves could be observed. The transgenic lines were evaluated for dehydration stress tolerance by germinating the nontransgenics and transgenics on 0.3 M and 0.4 M mannitol. Only 60% of nontransgenics germinated while all the transgenics with the exception of one line showed » 90% germination. The OSISAP1 overexpressing lines were also analyzed for tolerance to salt stress. Salt stress was given for 4 days and the difference between the fresh weight of nontransgenics and transgenics was recorded on day 8 of recovery. The transgenics performed better both in terms of fresh weight and retention of green color³.

The exact mechanism of how OSISAP1 overexpression confers tolerance to different abiotic stresses remains to be worked out. Several other stress responsive genes with unknown function have been reported to confer tolerance to plants upon overexpression. Overexpression of COR15a from Arabidopsis leads to increased freezing tolerance⁴. Similarly, HVA1 from barley was overexpressed in rice and found to confer stress tolerance⁵. Like COR15a and HVA1, OSISAP1 is also a hydrophilic protein that may contribute an increased tolerance to abiotic stress. It may also act as a signal cascade component using the zinc fingers for protein-protein or protein-DNA interactions. Furthermore, OSISAP1 shows homology to mammalian A20 protein. The mammalian counterpart has been shown to inhibit tumor necrosis factor-induced apoptosis through inhibition of NF-kappa B mediated gene expression⁶. Thus, it could be possible that OSISAP1 overexpression leads to less stress-associated injuries like chlorosis and cell death, and hence the transgenics performed better than the nontransgenics.

OSISAP1 could be a promising target for producing stress tolerant crops because it is inducible by different kinds of abiotic stresses and, upon ectopic overexpression, the transgenics show improved tolerance to cold, dehydration, and salt stress. It is quite possible that the gene may have a role in imparting tolerance to other stresses, which causes an increase in its transcription in rice.

1. Chinnusamy V, Schumaker K, & Zhu J-K. (2004) Molecular genetic perspectives on cross-talk and specificity in abiotic stress signaling in plants. J. Exp. Bot. 55:225-236.

2. Khush GS. (1997) Origin, dispersal, cultivation and variation of rice. Plant Mol. Biol. 35, 25-34.

3. Mukhopadhyay A, Vij S, & Tyagi AK. (2004) Overexpression of a zinc-finger protein gene from rice confers tolerance to cold, dehydration, and salt stress in transgenic tobacco. Proc. Natl. Acad. Sci. USA 101:6309-6314.

4. Artus NN et al. (1996) Constitutive expression of the cold-regulated Arabidopsis thaliana COR15a gene affects both chloroplast and protoplast freezing tolerance. Proc. Natl. Acad. Sci. USA 93:13404-13409.

5. Xu D et al. (1996) Expression of a late embryogenesis abundant protein gene, HVA1, from barley confers tolerance to water deficit and salt stress in transgenic rice. Plant Physiol. 110: 249-257.

6. De Valck D et al. (1997) A20 inhibits NF-kappaB activation independently of binding to 14-3-3 proteins. Biophys. Biochem. Res. Commun. 238: 590-594.

The green revolution of the 1960s introduced the production of short-stemmed wheat and rice varieties that were higher yielding compared to their taller counterparts. This development transformed agriculture, and perhaps signaled the realization of genetic crop improvement. Since then, scientists have continued working to understand crop genetics and physiology in order to refine strategies of manipulating crop growth. It is known that the green revolution wheat varieties are short because they respond abnormally to the plant growth hormone gibberellin (GA), an endogenous regulator of plant growth¹. GA signaling is mediated by a class of proteins called DELLA proteins, characterized by a region of 17 amino acids known as the DELLA domain. The best understood of these proteins are GAI and RGA. DELLA proteins repress growth, and their activity is opposed by GA.

Most of what we know about the role of these proteins in plant growth regulation comes from Nicholas Harberd and his research group at the John Innes Center in the UK, who have been investigating DELLA proteins for a number of years. In 1997, Harberd's group reported cloning the Arabidopsis GAI (Arabidopsis Gibberellin Insensitive) gene, encoding the first described member of the DELLA protein family². The gai gene encodes a mutant protein, gai, lacking the DELLA domain. Deletion of this domain causes reduced GA responses and dwarfism. Knowing the dwarf varieties of the green revolution were due to alterations in GA-mediated plant growth regulation, led to the realization that gai can be used to control crop plant architecture. This effect was demonstrated in rice³ in which transgenic plants containing a mutant GAI allele gave reduced responses to gibberellin and were dwarfed, indicating that mutant GAI orthologues could be used to increase yield in a wide range of crop species.

The constitutive expression of gai, however, could likely confer some undesirable phenotypes, and thus may not be desirable for crop improvement. For example, the rice obtained from the transgenic expression of GAI³ had a reduced ability to extrude the rice panicles completely from the surrounding leaf sheaths. This result might negatively affect yield, counteracting the benefits of the shorter stem size. An alternative mechanism is required for controlling growth when temporal and spatial regulation is needed; that is, imagine the ability to walk into a cornfield and turn off a growth switch so that the plant ceases to grow tall at a particular developmental stage.

In a recent publication in the Plant Biotechnology journal⁴, Harberd's group report ethanol inducible gai expression, using the ethanol inducible promoter AlcA. The "switch" in this case is ethanol and the repression of growth is dose-responsive. The dose-response effect means that instead of an on and off switch, the researchers have use of something akin to the volume knob on a radio. Therefore, they not only can ask the plant to dwarf or not to dwarf, but when to dwarf and by how much. Using Arabidopsis as their model, Harberd's team showed that the growth of plants transformed with the AlcA:gai construct was restrained by ethanol treatment, with the growth restraint due to the inhibition of GA response. They propose that this system could be used to tailor the growth properties of a variety of different crops to increase harvest index.

DELLA proteins have also been found in plants other than Arabidopsis, and they have been shown to mediate GA-responses in all the plants that contain them. In this system, the gai protein can be induced to act as a growth repressor at various stages of growth of AlcA:gai plants, and the effect on plant form depends on the developmental stage at which induction (by addition of ethanol) is effected. This suggests that gai affects plant parts that are actively growing at the time of induction, an observation consistent with the fact that GA regulates plant growth by affecting cell proliferation in young expanding organs. This and preceding work on these proteins have also provided further evidence for conservation of gene function from dicots to monocots, because the Arabidopsis dwarf gai gene transformed into rice confers the dwarf phenotype.

"Switchable" expression systems may obviate some of the environmental concerns regarding genetically modified crops. They allow researchers to regulate gene expression at a particular developmental stage, in a specific tissue or organ, and for a specified duration. Ethanol is also viewed as an environmentally-friendly "green inducer." Other chemically-inducible systems include tetracycline-, steroid-, copper-, and insecticide-inducible systems. Outside agriculture, these systems are helping to answer major questions in biology and evolution. A common method used in searching for primary targets of a transcription factor is to fuse it to something such as the glucocorticoid receptor, which can be induced with dexamethasone or cyclohexamide to suppress de novo protein synthesis.

1. Peng J et al. (1999) `Green revolution' genes encode mutant gibberellin response modulators. Nature 400: 256-261.

2. Peng J et al. (1997) The Arabidopsis GAI gene defines a signaling pathway that negatively regulates gibberellin responses. Genes Dev. 11: 3194-3205.

3. Fu X et al. (2001) Expression of Arabidopsis GAI in transgenic rice represses multiple gibberellin responses. Plant Cell 13: 1791-1802.

4. Ait-ali T, Rands C, and Harberd NP. (2003) Flexible control of plant architecture and yield via switchable expression of Arabidopsis gai. Plant Biotechnology Journal  1(5): 337-343.

A promising protein production system is the hen oviduct. Hen egg white is a relatively simple mixture, composed of approximately eleven major proteins, thus facilitating product purification. The major egg white proteins are found in milligram to gram quantities per egg, and almost all are glycosylated. Pathways for both N- and O-linked glycosylation are highly active in the tubular gland cells of the oviduct, which secrete egg white proteins. Manufacturing processes for egg cleaning, cracking, and protein purification are already in place for several companies on industrial scales. A modern hen produces more than 300 eggs per year, and the relatively short time to sexual maturity (~5 months) for chickens means that a transgenic flock with desired characteristics can be expanded rapidly.

To demonstrate proof-of-concept for the hen oviduct as a bioreactor for pharmaceutical proteins, we generated transgenic hens that express human interferon a-2b (hIFN) in their egg white¹. hIFN is primarily used for hepatitis C treatment protocols and represents a greater than $2 billion per year market². The resulting egg white-produced hIFN was biologically active in cultured cells, and glycosylation analysis revealed structures that matched the glycosylation pattern of interferon made naturally by human cells.

Generation of transgenic chickens
To maximize expression of hIFN in the oviduct, we synthesized the hIFN gene using a codon-usage table compiled from the four most abundant egg white proteins (ovalbumin, lysozyme, ovomucoid, and ovotransferrin). In this codon-optimization strategy, cognate tRNAs for the respective codons are assumed to be not limiting in the tubular gland cells since they are used by the most abundant proteins found in egg white. The codon-optimized hIFN gene was synthesized by amplification of overlapping nucleotides, and then placed in an expression vector controlled by the cytomegalovirus immediate-early promoter.

To date, the only way to generate birds with germline inheritable foreign genes is by retroviral-mediated transgenesis³. Therefore, we chose a replication-defective avian leukosis virus expression vector to introduce the gene encoding hIFN into the chicken genome. A viral producing cell line containing the hIFN gene was generated, and transducing particles were injected into ~350 stage X chicken embryos.

Upon reaching sexual maturity, rooster sperm was screened for the presence of the transgene by real time PCR. Based on the amount of the transgene found in sperm DNA samples, one rooster was chosen to breed to nontransgenic hens in order to establish a founder (G₁) for the hIFN flock. Approximately 1600 offspring were screened by real time PCR to identify the one founder, chick #1910. Integration of the hIFN transgene was verified by Southern blot analysis, and the G₁ rooster was bred to nontransgenic hens to generate the 50 G₂ hens used for egg white studies.

Evaluation of transgenic hen egg white material
Egg white samples from the 50 G₂ hens were assayed in several ways. To quantitate hIFN levels, a sensitive sandwich ELISA was used. Quantitation of hIFN in egg white revealed that the 50 hens produced an average of 2.7 µg/ml. The hIFN sandwich ELISA was also used in a time course study of two hens to determine that this expression level was maintained for at least seven months duration.

Biological activity of the egg white-derived hIFN was tested in a viral inhibition assay using human lung carcinoma cells challenged with mengovirus. In this assay, the hIFN produced in egg white had similar viral inhibition activity as purified standard hIFN.

In order to compete with existing protein production platforms based on E. coli expression systems, the foreign proteins produced in egg white of transgenic hens must be properly glycosylated. For many pharmaceutical proteins such as monoclonal antibodies and erythropoietin, favorable pharmacokinetics are dependent on proper glycosylation. Therefore, hIFN was purified from egg white material by chromatography and subjected to carbohydrate structural analysis.

Carbohydrates of purified hIFN were analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE). FACE involves labeling carbohydrates with negatively charged fluorescent dyes by reductive amination followed by digestion with different combinations of various exoglycosidases. Products are then electrophoresed on polyacrylamide gels, and the migration patterns of bands on the gels reveal the identity of particular carbohydrate groups. Six major glycoforms from the purified hIFN were analyzed in this manner, and 38% of the egg white-derived hIFN was found to match two of the three natural hIFN glycoforms produced in humans. One of the natural hIFN glycoforms, a monosialyl-pentasaccharide, was not represented in the hIFN purified from transgenic hen egg white. Although the glycosylation pattern of the egg white-derived hIFN was not identical to that produced in humans, the majority of the hIFN was in fact glycosylated. Current production platforms such as CHO cell bioreactors also produce glycosylated proteins that do not exactly match the glycosylation structures of their natural human counterparts⁴. Further studies will determine the pharmacokinetics of the various glycoforms found in hIFN from egg white.

Future directions for the hen oviduct bioreactor
In conclusion, we have demonstrated that transgenic hens are capable of producing hIFN in egg white. The hIFN secreted in hen eggs is biologically active since it inhibits viral infection of cultured human cells. In addition, approximately one-third of the hIFN is glycosylated in the same manner as naturally produced hIFN. Further work will increase hIFN expression levels in hen egg white to make the hen oviduct bioreactor an economically feasible platform. In addition, genes for several different glycosylated pharmaceutical proteins will be introduced into the chicken genome to determine the general applicability of this novel production platform. Another area of research is to develop an alternative gene delivery platform that would allow much larger transgenes to be introduced into the chicken genome. Larger constructs would include matrix attachment regions to prevent gene silencing as well as introns and other regulatory elements to maximize expression of foreign proteins.

1. Rapp JC et al. (2003) Biologically active human interferon a-2b produced in the egg white of transgenic hens. Transgenic Research 12: 569-575.

GLOFISH, THE FIRST GM ANIMAL COMMERCIALIZED: PROFITS AMID CONTROVERSY
Eric Hallerman

The GloFish, a fluorescent red zebrafish sold as a novel pet, has become the first transgenic animal sold to U.S. consumers. Its sale has produced regulatory controversies, a lawsuit, and profits for its proponent, Yorktown Technologies (Austin, TX). With the market plan calling for sales in a widening number of countries, continuing controversy seems likely.

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What is a GloFish?
The GloFish is a trademarked transgenic zebrafish (Danio rerio) expressing a red fluorescent protein from a sea anemone under the transcriptional control of the promoter from the myosin light peptide 2 gene of zebrafish¹. Produced and patented by a group at the National University of Singapore, exclusive rights for international marketing were purchased by Yorktown Technologies approximately a year-and-a-half ago. Yorktown produces GloFish through contracts with 5-D Tropical (Plant City, FL) and Segrest Farms (Gibsonton, FL), and began marketing them in the United States in December.

Issues posed
The prospect of commercial sales of GloFish raised a number of issues. Among them was the issue of whether GloFish pose an environmental hazard. Zebrafish, a tropical species native to south Asia, are sensitive to low temperature. Despite decades of production and use in the U.S., zebrafish have not established self-sustaining populations within the country. Laboratory tests¹ showed that viability, reproductive success, and temperature tolerance of transgenics were equal to or somewhat less than those of the wild type. While preliminary, results supported the expectation that the modification would not increase invasiveness, and that environmental risk was small.

Commercialization of the GloFish in the United States poses regulatory uncertainty because existing biotechnology policy bases oversight on the use of the product. Sales of ornamental fishes are not federally regulated. The Food and Drug Administration asserts jurisdiction over genetically modified animals using the New Animal Drug Application process. After a brief internal review and interagency consultation, FDA's Center for Veterinary Medicine determined that "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 any clear risk to the public health, the FDA finds no reason to regulate these particular fish²." Alan Blake, CEO of Yorktown Technologies, also made contact with the U.S. Department of Agriculture, the U.S. Fish and Wildlife Service, and the Environmental Protection Agency, which expressed no regulatory concerns regarding GloFish.

States generally have lead authority on fisheries issues. California is the only state that forbids the possession, sale, and transport of genetically modified fishes. Yorktown asked for an exemption, but on December 3, the California Fish and Game Commission voted not to approve sale of the fish. Explaining the decision, Commissioner Sam Schuchat wrote, "Creating a novelty pet is a frivolous use of this technology. No matter how low the risk is, there needs to be a public benefit that is higher than this³." Many California consumers were unhappy with the Commission's decision. Following appointment of a new commissioner and a change of mind by a continuing commissioner, on April 1 the Fish and Game Commission reversed their earlier decision by a 3-1 vote⁴. They asked for an updated recommendation from the Department of Fish and Game and will hold public hearings before making a final decision. The final vote of the Commission is expected near the end of June. If approved, GloFish would become available for sale in California by the end of July.

Commercialization of GloFish sparked legal action to force the FDA to exert regulatory oversight and block further sales. On January 14, the Center for Technology Assessment (CTA) and Center for Food Safety (CFS) filed a lawsuit in the Federal District Court in Washington, D.C. challenging FDA's implementation of the Federal Food, Drug, and Cosmetics Act (FFDCA), National Environmental Policy Act (NEPA), and Administrative Procedures Act regarding procedures by which FDA allowed commercialization of GloFish⁵. CTA and CFS allege that FDA inaction poses harm to the enjoyment of natural waters, harm to carnivorous fishes, increased exposure to antibiotic-resistant bacteria and viruses due to elements in the expression vector used to produce GloFish, and aesthetic injury from viewing genetically engineered GloFish in aquaria. CTA and CFS asked the court to declare that FDA has regulatory authority under FFDCA, "absence of a clear risk to public health" is not the appropriate regulatory standard, and FDA action was not compliant with NEPA, and to enjoin FDA from allowing further sales of GloFish. The lawsuit is currently pending.

Future prospects
Future prospects for the GloFish include marketing additional color lines in a wider range of markets. Not only red, but also green and yellow fluorescent proteins have been introduced into stable transgenic lines, yielding green, yellow, and orange fish.

Commercialization of fluorescent zebrafish has gone forward in several countries and is stymied in others. Fluorescent green zebrafish developed in Taiwan have been sold in Taiwan, Malaysia, and Hong Kong. Singapore confiscated attempted imports of the fish. Despite this, Yorktown Technologies is considering other markets, including parts of Asia and Latin America. Extensive information requirements suggest that GloFish will not be marketed in Canada or the European Union in the near future. Despite these regulatory challenges, according to Blake, "The GloFish venture is a profitable one, and the company looks forward to continuing to provide a safe and enjoyable product for many years to come."

Issues posed by commercialization of GloFish will be with us for years to come. Approval for commercializing other transgenic fishes, including Atlantic salmon expressing an introduced growth hormone (GH) gene, has been sought. GH-transgenic tilapia, channel catfish, and rainbow trout are on the regulatory horizon, and a wide range of other transgenic fishes are under development. Marketing of fluorescent zebrafish may spur efforts to develop other transgenic ornamentals, such as goldfish and koi carp. Transgenic zebrafishes also are under development for biomoni-toring—expression of fluorescent protein genes under the control of estrogen-inducible or stress response promoters would indicate exposure of caged zebrafish to environmental pollution⁶.

1. Gong W et al. (2003) Development of transgenic fish for ornamental and bioreactor by strong expression of fluorescent proteins in the skeletal muscle. Biochemical and Biophysical Research Communications 308: 58-63.

Golf course managers may soon have a new weapon for their battle against weeds like annual bluegrass: creeping bentgrass (Agrostis stolonifera) genetically modified (GM) for tolerance to Roundup^®, a glyphosate herbicide. In April 2003, Monsanto Company and The Scotts Company filed a petition with the U.S. Department of Agriculture's Animal Plant and Health Inspection Service (APHIS) seeking commercial approval for Roundup Ready^® Creeping Bentgrass. The companies plan to sell the GM grass for use in commercial grass seed production and, ultimately, for golf courses.

In their petition, the companies argue that Roundup is highly effective against the majority of annual and perennial grasses as well as broadleaf weeds common to grass and turf production. They also claim that the herbicide has excellent environmental features, such as rapid soil binding and low toxicity to mammals, birds, and fish. In addition, they argue that glyphosate is one of the few herbicidal active ingredients that the Environmental Protection Agency has classified as having evidence of non-carcinogenicity for humans. The GM bentgrass has been under development since 1998 and has been field tested in a number of states.

APHIS opened a 60-day period for public comment on the petition in early 2004. By the time the comment period closed in March, APHIS recorded over 450 remarks. Among these was a comment from the Union of Concerned Scientists who noted that the GM grass is unlike typical genetically engineered crops under regulatory review. Annual crops depend to varying extents on humans for successful propagation. In contrast, GM creeping bentgrass is a perennial that establishes without cultivation in a wide variety of habitats and reproduces both sexually through seeds and vegetatively by horizontal stems that produce roots. Bentgrass is also wind-pollinated.

In its preliminary risk assessment, APHIS noted that creeping bentgrass could form hybrids with at least 13 other U.S. naturalized or native species of grass. The Union of Concerned Scientists warns that, if deregulated, GM creeping bentgrass could be planted within pollinating distance of these compatible relatives and this could lead to the establishment of transgenes in wild plants. The organization urges the USDA to delay a decision on the bent-grass petition until the agency completes its repromulgation of regulations governing GM plants under the Plant Protection Act of 2000. Representatives from the Bureau of Land Management, the U.S. Forest Service, and the U.S. Army Corps of Engineers have also expressed concerns about GM bentgrass.

After reviewing and evaluating comments on the petition, APHIS will draft a document under the National Environmental Policy Act to assess the potential environmental impacts of a decision to deregulate the product. The environmental document will be made available for public comment. APHIS will make its decision about the petition after reviewing this second round of remarks.