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ENABLING SCIENCE Antifreeze proteins and their genes: From basic research to business opportunityIncorporating AFPs at the transgenic and product levels offers the potential to boost profits in the biotechnology, food, and agricultural industries.
Garth L. Fletcher
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-helices found in righteye flounder (Pleuronectidae) and in shorthorn sculpin (Myoxocephalus scorpius). Type II AFPs, characterized by disulfide bridges and an extensive 
-structure, are found in sea ravens, smelts, and herring. Type III AFPs are compact 
Table 1.The principal function of AFPs in fish is to lower the freezing temperature of their blood and extracellular fluids, thus affording protection from freezing in ice-laden marine waters. The freezing points of seawater (-1.8 °C approx.) and the plasma of six teleost (bony fish) species (3, 6-8) of Atlantic Canada are compared in Figure 1 (below). Experiments show that fishes with no antifreeze protection freeze at temperatures considerably higher than -1.8 °C, whereas species that produce AFPs are able to survive in low-temperature seawater because of their high plasma antifreeze levels.
Figure 1.The capacity of a species to produce antifreeze protein (AFP) reflects the severity of the species' overwintering environment. When 5 mg/mL AFP Type I was injected into rainbow trout (a species that does not normally produce AFPs), we found that freeze resistance was enhanced. This suggests that salmonids transgenic for AFP could benefit from the presence of plasma AFP and be more resistant to cold than their nontransgenic relatives.
AFPs serve as antifreeze agents, not by acting colligatively as would most solutes (e.g., electrolytes) but by specifically adsorbing to the surface of ice crystals as they form, thereby preventing their growth. Because of unique aspects of their tertiary structures, these proteins are up to 500 times more effective at lowering the freezing temperature than any other known solute molecule. Thus, teleost fishes have evolved a mechanism to reduce the freezing point of their bodily fluids without appreciably changing their osmolarity (2, 3). The evolution of these AFPs and their genes has been reviewed (10, 11).
In this article, we focus on examples for which there is a market for an antifreeze product and where the scientific basis for such an application has been established or is reasonably well understood. Because commercial applications of these proteins are still in the R&D phase, the economic viability of such ventures must be left to future evaluation. Two excellent reviews provide additional details about the potential uses of AFPs in food products (12, 13).
On the basis of our current knowledge about AFPs and AFP genes, commercial applications have been identified in the following areas (Table 2) (14): protection of fish and plants against cold and freezing temperatures;
- cold protection of mammalian cells, tissues, and organs;
- enhanced tumor cell destruction during cryosurgery;
- longer shelf life for and better quality of frozen foods; and
- improved growth characteristics in transgenic fish by using AFP gene promoters.
Table 2.
Potential uses of AFPs in aquaculture
In terms of aquaculture sites, Canada's east coast is frequently compared with the coast of Scandinavia; however, the prevailing ocean currents in these two areas are very different. Whereas water temperatures are relatively mild along the coasts of Norway and Sweden year-round, the marine environment along much of Canada's Atlantic coast is characterized by ice and subzero water temperatures (0 to -1. °C) during the winter (Figure 2). Such an environment is lethal to most teleost fishes; the primary exceptions are those that can produce AFPs. Salmonids and many other commercially important fish cannot synthesize antifreeze; they freeze and die when they come into contact with ice crystals at temperatures lower than -0.7 to -0.9 °C. Thus, although many locations in eastern Canada appear suitable for salmon grow-out operations, most of them cannot be used with existing technology because temperatures drop below -0.7 °C (15).
Because of these environmental factors, the marine cage culture of salmonids in eastern Canada is almost entirely restricted to the southern New Brunswick in the vicinity of Passamaquoddy Bay (Figure 2), where the waters freeze only infrequently (16). However, even at this location, where the value of the aquaculture industry is about $100 million annually, the danger that water temperatures could decline to lethal levels remains, and mortalities attributed to "superchill" are not uncommon (17, 18). Loss of salmon as a result of superchill thus severely restricts aquaculture along the coast of eastern Canada, and it can be a problem for salmonid aquaculture even as far south as Maine.
Figure 2.The only failsafe method to ensure that sea cage cultured salmon will not die of superchill during the winter is to improve their freeze resistance. One route is to modify their genomes by adding AFP genes. Antifreeze proteins introduced into the circulation can improve the freeze resistance of salmonids with no prior adaptation or specialization (9 ). We reasoned that transgenic salmonids with the AFP gene would be able to produce endogenous AFPs and thus increase their freeze resistance. We started testing this concept in 1982 by microinjecting fertilized salmon eggs with 1 million copies of an AFP gene from winter flounder (5). The results of these experiments demonstrated that the antifreeze transgene was successfully integrated into the salmon chromosomes, expressed, and, to date, has exhibited Mendelian inheritance through five successive generations (19-21).
Our studies with antifreeze gene transfer demonstrated conclusively that transgenes can be incorporated into salmon chromosomes, expressed appropriately, and passed from generation to generation in a predictable Mendelian manner. We proved that salmon and other fish species can be rendered more resistant to freezing by gene transfer. The challenge now is to redesign the antifreeze gene construct to include a strong promoter/enhancer coupled to a gene for a more powerful AFP. For example, the highly active AFPs discovered in insects (22) could serve as a basis from which to design a more potent antifreeze gene for use in fish. This problem is regarded as technical rather than conceptual. Therefore, a solution can be expected within a reasonable time.
The time and effort required to develop freeze-resistant fish for aquaculture is considerable; however, the economic benefits to the Atlantic region of such a breakthrough could be significant. Not only would the danger of superchill mortalities be eliminated at aquaculture sites currently in use, but sea cage aquaculture could be extended to numerous locations along the Atlantic coast where the only barrier is the low water temperature.
Crop protection with AFPs
The damaging effects of low temperatures and freezing conditions on plant material consist of mechanical injury (cell and tissue disruption), which results from ice formation, and dehydration injury caused by water loss associated with ice formation. Under deep-freezing conditions, intracellular bulk water and water oriented on the surface of macromolecules and on the polar heads of lipids in cellular membranes are effectively removed, causing severe dehydration and structural and functional damage to plasma membranes (23, 24).
Although many plants are killed by low and freezing temperatures, some species and varieties have developed physiological processes to help them survive the winter. With the onset of cold conditions, some plants produce colligative cryoprotectants such as sucrose and proline; in others, changes in membrane lipids and proteins that render membranes more cold stable have been reported (23). A few plants are able to produce cold-regulated cryoprotective proteins or AFPs, which protect them from cold damage under freezing conditions (25-29).
AFPs similar to those found within the animal kingdom have been identified in plants (12, 30) and appear to behave similarly at freezing temperatures (30). In addition to their protective action at subzero temperatures, fish AFPs protect cold-sensitive cells at hypothermic temperatures, possibly by interacting with the cell membrane (31). A similar effect may be exerted by plant cold protective or antifreeze proteins in that they may provide a protective environment in the proximity of the membranes that stabilizes the membrane or inhibits ice propagation through cell walls (27, 32).
At least 30 species of angiosperms are capable of antifreeze activity after they become acclimated to low temperature, and antifreeze activity has been found in commercially grown species - winter rye, spring and winter wheat, winter barley, spring oat, winter canola, potato, carrot, cabbage, kale, and brussels sprouts (12). However, levels are generally low, and the majority of crops grown commercially do not possess such defense mechanisms.
A surprising number of valuable plant species succumb to frost damage each year, with concomitant hardship to growers and consumers (particularly in developing countries). Some crops that have made the headlines in recent years because of devastating frost damage - and hence lost revenues - are wheat (33, 34), coffee, soybeans, alfalfa, tobacco, potatoes, sugar beet, corn, rapeseed, safflower, citrus, strawberries, cherries, apricots, peaches, pears, and apples (33, 34). Colorado's fruit-growing areas regularly lose a percentage of fruit crops (cherries, apricots, peaches, pears, and apples) to frost; 1998 (an El Niño year) was the first year in nine years that frost did not damage the crops (35). However, in California, toward the end of 1998, freezing conditions resulted in citrus fruit losses valued in excess of $634 million (36). In 1997, frost damage to wheat in Oklahoma, Texas, and Kansas was reported as severe (37). Considerable losses of strawberries and raspberries were experienced in Ireland the spring of 1998 (38). Soft fruit crops are particularly susceptible to frost, and planting regimes that maximize the growing season may actually increase the danger of frost damage.
Exact figures for loss of revenue due to frost damage are difficult to obtain because crop values fluctuate, and the losses can only be estimated. However, a few statistics can give an idea of the scale of losses. In parts of the corn belt, soybeans valued in excess of $500 million were lost in 1995 (39). Brazil is subject to recurrent lost revenue due to the impact of frost on its coffee crop; estimated 1994 crop losses were at least $2 billion (40). Shortfalls in coffee supply are a regular occurrence due to frost damage; the latest was in 1997 (41). In March 1998, a frost in Georgia caused the loss of soft fruit (blueberries and strawberries), peach trees, and other crops estimated at approximately $200 million (42). In 1996, frost damage to the Florida citrus crop was estimated at $360 million (43).
With the increased popularity of exotic fruits and their cultivation in nontraditional areas, it is necessary to produce hardy cultivars that are not susceptible to frost damage. One example of this is the kiwifruit (44). Since 1985, kiwifruit growers in British Columbia, Canada, have sustained substantial losses due to frost. More recently, kiwifruit growers in France, Italy, New Zealand, California, and South Carolina also have experienced losses. For nontraditional crops such as aloe vera or imported ornamental plants of nontraditional origin, even short temperature drops can be devastating (45).
It would be desirable to increase the cold hardiness of plants grown in areas susceptible to frost, but how might this be achieved?
Producing cold-hardy varieties. Although the use of traditional breeding programs to develop cultivars of certain commercially important crops with improved low-temperature tolerance is ongoing, progress so far has been slow (46). The ability of plants to withstand low temperatures appears to be a function of three separate heritable traits: freezing tolerance, freezing avoidance, and speed of acclimation (46). Although some improvements in cold hardiness have been achieved (47), understanding the control of these traits and their manipulation at the genome level is a vast undertaking and is still in the early stages for most crop species (48, 49). Whether the lack of speed is due to the difficulty in identifying the appropriate genetic markers (46), the limitation of naturally occurring genetic variability in cold-hardy traits (48), or other factors such as the vagaries of the environment during selection trials, the desired level of cold tolerance needs to be determined for commercially important crops, increased cold tolerance is still required in commercially important crops.
In recent years, with the advent of transgenic technology, effort has been directed toward enhancing the gene complement of crop species with novel genetic material. AFP genes are now being introduced into plant and animal species to extend their geographic range, increase their chances of overwintering survival, and improve the texture of products that might be stored frozen after harvest.
In one experiment, leaves of potato, canola, and Arabidopis thaliana plants were vacuum infiltrated with AFP Type I from winter flounder (50). The experimental plants and the control (water-infiltrated) plants of the same three species then were exposed to freezing conditions. The AFP-infiltrated plants were found to be more cold-hardy than the controls. The experimenters found that this method of AFP introduction depressed the freezing point to a level that would significantly improve crop survival under agricultural conditions.
The use of transgenic technology to produce plants capable of synthesizing their own antifreeze has yielded transgenic tobacco, tomatoes, and potatoes (29, 51-53). In these transgenic plants, researchers have observed increased freeze resistance and inhibition of electrolyte leakage from cold-stressed cells.
Depending on the role the AFPs are expected to play in transgenic plants, the levels of antifreeze expression required will differ. For example, higher levels (~1000-fold) are needed to inhibit ice crystal formation and propagation than are needed to protect frozen products from ice recrystallization during storage (53). Consideration must be given to the type of antifreeze molecule best suited to the recipient organism. In plant systems, plant antifreeze or synthetic molecules may be more acceptable to plants, whereas fish antifreeze molecules would seem to be more acceptable in fish species (12). There is plenty of scope for fine-tuning this technology to arrive at the most appropriate kind of molecule and level of expression. On the basis of the magnitude of crop losses due to frost each year and the progressive adoption of exotic (and less hardy) species in the Western diet, the demand for increased cold tolerance can only grow in the coming years.
Low-temperature preservation of cells, tissues, and Organs
Until 1990, it was generally believed that the sole function of AFPs was to protect fish from freezing; however, in a series of experiments published after 1990, Rubinsky and colleagues discovered that all of the antifreeze types known at that time could improve the cold tolerance of cold-sensitive mammalian cells (54). In initial experiments, the researchers incubated bovine oocytes at 4 °C for 24 h in the presence and absence of AFPs, then rewarmed both the test and control cells. Oocytes stored cold in the presence of AFPs were as capable of normal maturation, fertilization, and embryonic development as were freshly collected oocytes; however, oocytes stored cold without AFPs lost their membrane integrity and died (Figure 3) (55, 56). Additional experiments extended these observations by demonstrating that AFPs could help protect functional aspects of whole rat livers following hypothermic (lower than normal temperature, nonfrozen) cryogenic (very low temperature, frozen) storage (57, 58
).
Figure 3.Precisely how AFPs help maintain cell membrane integrity at hypothermic temperatures remains unknown. Results from experiments carried out to date suggest two distinctly different hypotheses. One hypothesis for cell death in cold-sensitive animals involves the alterations that occur in membrane and cellular functions at low temperatures (59, 60). Exposure to low, nonfreezing temperatures reduces the rate of cellular metabolic processes to the point at which they are unable to produce sufficient energy to maintain the cell membrane ion pumps. However, because passive ion flow through cell membrane channels is largely unaffected by temperature, the inability of the ion pumps to function effectively leads to membrane depolarization, increased intracellular Ca2+ concentration, activation of membrane phospholipid hydrolysis, and, finally, cell death. Experiments that demonstrated that AFPs could block passive Ca2+ entry into rabbit parietal cells support this hypothesis and suggest that the protective effect of the AFPs may be related to their ability to block the entry of Ca2+ into the cell (61).
A second hypothesis, apparently unrelated to the ion channel-blocking action of the AFPs, is their observed ability to protect cell membranes as they pass through the phase transition temperature. When cells are cooled through the phase transition temperature of their lipid membranes, they undergo a phase change from liquid crystal to gel. Consequently, the membranes become transiently leaky, and intracellular contents are lost. Hays and co-workers used dielaidoylphosphatidylcholine (DEPC) liposomes as a membrane model, it was found that AFGPs can protect the integrity of the lipid bilayers, thereby inhibiting the leakage of liposome contents during the thermotropic phase transition
(Figure 4) (62
).
Figure 4.Following the studies in liposomes (62 ), AFGPs were tested for their ability to protect blood platelets from activation at low temperatures. When platelets are exposed to low temperatures for even a short time, they release their contents through a process known as cold activation. Platelet cold activation is thought to be related to passage through the phase transition temperature, with resulting membrane leakiness. Platelet activation is initiated during phase transition, and once started, the process appears to continue to completion, leading to total loss of platelet function.
The inability of platelets to retain function at 4 °C has been a stumbling block to their efficient cold storage for transfusion and is the cause of considerable platelet loss. The current practice of storing platelets at 22 °C limits their shelf life to 5 days, after which they are prone to bacterial contamination and activation and must be discarded (63). Experiments showed that AFGP was able to protect platelets cooled to 4 °C and held at this temperature for 21 days (Figure 5) (63 ). The exact mechanism by which this protective effect is exerted has yet to be determined; however, it seems likely that inhibition of platelet membrane leakage during and after phase transition, possibly coupled with inhibition of Ca2+ leakage from the dense tubular system of membranes within the platelet, is involved (63).
Figure 5.Most studies that evaluate the efficacy of AFPs in preservation media have concentrated on the hypothermic or cryogenic storage of reproductive materials. Improved long-term storage techniques for such cells would have many commercial applications in the fields of agriculture (e.g., in vitro fertilization techniques, development of improved blood lines), aquaculture, and human reproductive technologies.
Improved protocols for extended hypothermic or cryogenic storage of human organs intended for transplantation would be a considerable advance in transplant technology. At present, the storage time limits for transplant hearts and livers are ~6 and 12 h, respectively, at 4 °C. Because of the huge demand for human transplant tissues and organs, any technology that improves the quality and shelf life of stored material would have a significant effect in medical and financial terms.
Studies demonstrating the protective effects of AFPs on cells and tissues at cryogenic and hypothermic temperatures are presented in Table 3 (55, 57, 63-71). However, not all studies have found that AFPs confer cold protection (72). Indeed, when used in cryogenic applications, AFPs, under certain conditions, can destroy cells.
Table 3.Promising studies have indicated that AFPs can protect cells at hypothermic and cryogenic temperatures. However, if AFPs are to meet expectations for profitable commercial use, more research is needed to tailor applications to the specific cell types, tissues, and organs that require protection. Details that need attention include AFP type, concentration, and purity; synergistic/antagonistic interactions with buffers and cryoprotectants; and thermal regimes during cooling and warming.
Our lack of detailed knowledge about how AFPs are acting in these applications is impeding progress toward the development of AFP products for the cold storage of cells, tissues, organs, and possibly whole organisms. The generation of a model that describes the nature of the molecular interaction between AFPs and cell membranes would help focus research on critical issues rather than the "try and see" approach evident to date.
More effective cryosurgery
Cryosurgery is a procedure in which probes cooled to very low temperatures are used to freeze and destroy solid tumors. The procedure is attractive to surgeons and patients alike because it is minimally invasive. The use of cryosurgery to destroy deep body tumors, such as those of the prostate and liver, has increased in recent years because of developments in intraoperative imaging systems that provide surgeons with real-time details on the extent of freezing within the tissue (73).
One of the problems with cryosurgery as it is currently practiced is the variation in its effectiveness in destroying tumor cells. Freezing does not guarantee cell death, as is evident from cryopreservation studies that have established that the life or death of a cell depends on its thermal experience during the freeze/thaw process. The number of target cells that survive cryosurgery is highly variable and depends on the thermal parameters used during surgery (e.g., rate of cooling, number of freezing cycles, and final freezing temperature before rewarming) (74 ). Inappropriate thermal parameters during the procedure are believed to be responsible, at least in part, for the variations seen in the clinical outcome (75 ). Because of the nature of the cryosurgical procedure, it may be difficult, if not impossible, to control the freeze-thaw thermal parameters accurately. A cryosurgical method that ensures the destruction of all frozen cells regardless of the thermal regime is required (75 ).
One potential solution to the problems associated with the efficacy of current cryosurgical practice lies with the manner in which AFPs interact with ice crystals and modify their morphology and growth. In a dilute solution, AFPs adsorb onto the prism faces and prevent or limit growth along the preferred a-axis. When the temperature of the solution is lowered appropriately, the crystal grows rapidly along the c-axis, resulting in a bipyramidal crystal. Crystals grown in high concentrations (5-10 mg/mL) of antifreeze are long and needlelike.
Koushafar and Rubinsky (75 ) took advantage of the ability of AFPs to produce needlelike ice growth by examining their effects on cultured human primary prostatic adenocarcinoma cells (ND-1) after freezing. The researchers observed that in the absence of AFP Type 1, a high proportion of the target cells survived freezing. However, when frozen in a 10 mg/mL solution of AFPs, all the cells were destroyed, regardless of the cooling rates used during freezing (Figure 6). Similar results were obtained in a follow-up study in which intact rat livers were perfused with AFPs (73 ). Therefore, it is evident that AFPs can serve as effective chemical adjuvants to cryosurgery by greatly increasing the effectiveness of the procedure at destroying solid tissue masses such as tumors.
Figure 6.
Improved storage of frozen foods
It is well known that some foods (e.g., strawberries, raspberries, and tomatoes) cannot be frozen without loss of quality caused by cellular destruction; even food products that freeze well deteriorate to varying degrees over time. One of the causes of deterioration during frozen storage is the growth of large ice crystals within the product when refrigeration temperatures are not optimal. This process, known as ice recrystallization, occurs when the products are stored at subzero temperatures or are subject to fluctuating subzero temperatures such as occur during freezer defrost cycles.
Recrystallization in frozen foods can result in two distinct problems:
- loss of the smooth creamy texture of frozen desserts (e.g., ice cream) and
- increase in the size of intracellular spaces, resulting in membrane damage and (once the product is thawed) gapping, reduced water-holding capacity (drip loss), and subsequent loss of nutrients.
Food quality and texture would be improved if a means could be found to reduce or eliminate this phenomenon.
AFPs are highly effective at inhibiting ice recrystallization at low concentrations (<0.1 µg/mL), an effect clearly illustrated in Figure 7 (76, 77 ). Initial crystal size was smaller and crystal growth was significantly retarded in the presence of AFGP; both parameters were negatively correlated with AFGP concentration. Thus, they could be used to help eliminate the destructive effects of ice recrystallization in frozen foods.
Figure 7.Payne et al. (78) were the first to investigate the potential usefulness of AFPs in preserving frozen meat quality by soaking bovine and ovine muscle in AFP-containing solutions before freezing and storage at -20 °C. They found that the antifreeze-treated samples had many small intracellular spaces within the muscle fibers, whereas untreated (control) samples had a smaller number of considerably larger spaces that produced a more spongelike appearance (Figure 8). Antifreeze addition prevented the formation of such ice crystals; only small ice crystals formed throughout the sample. Ice crystal size decreased with increased antifreeze concentration (from 1 ng to 1 mg) and length of soaking (from several hours to 7 days). A follow-up study, in which lambs were injected with AFPs before slaughter, showed similar findings (79). Sensory (e.g., taste, smell, appearance) analyses of frozen and thawed Antarctic cod revealed a lower drip loss and greater overall acceptability to the test panel than muscle from a closely related species of black cod (80 ). Because Antarctic cod naturally contain AFPs, these results imply that the better flesh quality could have been due, at least in part, to the presence of antifreeze.
Figure 8.One obvious impediment to using AFPs in whole meat or fish products is the difficulty in getting them into the product in the first place. It seems unlikely that AFPs will be administered to living animals before slaughter. Therefore, apart from developing a range of transgenic plants and animals that produce their own AFPs, applications are most likely to be in products such as frozen desserts or ground meats, in which AFPs can be readily mixed into the product before freezing.
Transgenic technology--a way to increase fish production
The world's fisheries are in danger of commercial extinction due to exploitation and overfishing (21, 81). Because fish is an essential protein source for the world population, it is critical that we develop alternative methods to ensure future quantities of fish. Aquaculture appears to be the only viable means of meeting the future demands for fish without driving one species after another to the brink of extinction (82).
A key element to enhanced production of cultured species is the development of genetically superior broodstocks that are tailored to their culture conditions and to the marketplace. Generally desirable characteristics include improved growth rates; feed conversion efficiency; resistance to disease; resistance to cold and freezing temperatures; tolerance of low oxygen levels; and the ability to thrive, when fed inexpensive, nonanimal (nonfish) protein diets (83).
Despite the acknowledged power of traditional selection and breeding methods (84), the development of superior broodstocks using this procedure is relatively slow, and in some cases, impossible. Therefore, to meet the needs of the 21st century, this process must be accelerated.
Transgenic technology provides the means to make quantum leaps in production. The identification, isolation, and construction of genes responsible for desirable traits and their transfer to broodstock fish present a powerful method of genetic and phenotypic improvement that would be difficult, if not impossible, to achieve by using traditional selection methods (85, 86).
We believe that there are two essential elements to the successful use of transgenic fish for food:
- The end product must be safe for the environment and for human consumption and
- recognized and acknowledged as safe, and therefore acceptable, to the public at large.
There is little point in spending the large sums of money and time required to produce transgenic fish if the products never could be marketed. It seems to us that the only reasonably safe approach to this problem is through the use of genes and promoters derived from fish rather than from humans, bacteria, or viruses.
All aquaculture ventures could benefit from the development of culture species with enhanced growth rates that would reduce the time required to raise fish to market size. Benefits would include increased productivity per unit of capitalization, in terms of sea cages or land-based units; a more rapid return on investment in livestock; reduced time for infection by pathogens; and reduced exposure to environmentally dangerous events such as predators, freezing waters, and algal blooms.
Growth hormone genes are normally expressed in the pituitary gland, where they come under the control of the central nervous system. To remove the central nervous system control, it is necessary to modify the tissue-specific elements of the growth hormone gene so that expression can take place elsewhere. Because tissue specificity generally lies within the promoter region, a chimeric growth hormone gene construct must use promoters that function in tissues other than the pituitary and, at minimum, include the following components: 5` flanking sequence (promoter sequence responsible for where the structural gene will be expressed, in what quantities, periodicity of expression, and what factors regulate expression, e.g., photoperiod); the candidate structural gene (cDNA or genomic DNA coding for a particular protein, e.g., salmon growth hormone); 3` flanking sequence (polyadenylation signal and transcription termination sequence, which controls termination of transcription and has an influence on mRNA stability) (Figure 9).
Figure 9.Antifreeze gene promoters are, in many ways, ideally suited to driving the expression of transgenes in fish. They are not present in commercially important species such as salmonids, halibut, tilapia (African freshwater cichlids), catfish, and carp; therefore, they are readily detectable by polymerase chain reaction in transgenic individuals because they cannot be confused with endogenous DNA sequences. The distinctly different antifreeze types available in nature provide a range of promoters to choose from. In addition, additional variety is provided by the fact that some of the genes are expressed on a seasonal basis, whereas others are expressed year-round; some are highly tissue-specific, and others are more ubiquitous in their expression (85, 87 ).
We developed an "all-fish" transgene cassette using an antifreeze gene promoter isolated from the ocean pout and linked to chinook salmon growth hormone cDNA (88 ) (Figure 10). The ocean pout promoter was chosen because it is expressed predominantly in the liver throughout the year (7, 89 ).
Figure 10.With the use of this all-fish growth hormone gene construct, we have now produced stable lines of transgenic Atlantic salmon that exhibit growth rates enhanced by four- to sixfold, making it possible to routinely produce 100-g smolts within five months of first feeding (20, 83, 90, 91 ). As a result, the time to grow Atlantic salmon from egg to market size in Atlantic Canada could be cut by a year if transgenic salmon were to replace standard salmon in aquaculture facilities. One example of the enhanced growth rates observed for F1 transgenics is presented in Figure 11.(below)
Figure 11.The scientific and technical components involved in the production of genetically engineered fishes for aquaculture represent only part of the effort that is required to integrate this technology successfully into industry. Of equal importance are all aspects of environmental risk assessment and considerations of animal welfare, food safety, intellectual property protection, and consumer acceptance ( 21 ).
Antifreeze protein sources
At present, relatively small amounts of AFP are being obtained for R&D, primarily from their most easily accessed natural source: the plasmas of cold ocean teleost fish, in which concentrations of 1-30 g/L have been reported (13
). However, as commercial applications are developed, the demand for greater amounts of AFP will increase. Clearly, the amount of AFP required will depend on the application.
How much protein can reasonably be obtained from a natural fish source? Right now, the answer to this question can be no more than a rough estimate. Factors such as fish species, time of year, plasma antifreeze concentration, and AFP losses that occur during purification affect the yield considerably. Therefore, the following estimates should be viewed with these variables in mind.
If the volume of plasma readily obtainable from fish is ~0.5% of the body weight, and 2 g of antifreeze can be extracted from each liter of plasma, then ~100 metric tons of fish would be required to produce 1 kg of purified antifreeze. Using this value as a basis for discussion, we can estimate the amount of fish required to supply some of the markets indicated in Table 2. For prostate cryosurgery, Rubinsky's evidence indicates that concentrations of 5-10 mg/mL would be necessary to assist in the destruction of the tumor cells; 500-1000 metric tons of fish per year would be required to obtain the necessary amount of AFP. In terms of a commercial fishery or an aquacultural venture, this amount of fish is not unreasonably large. However, in the case of a product such as ice cream, the picture is very different. For example, using a concentration of AFP that would be effective at inhibiting ice recrystallization (0.1 mg/L) (76), ~150,000 metric tons of fish would be required to obtain enough AFP for the projected market (Table 2). Clearly, it is foolish to think that either game or farmed fish could serve as a source of AFPs for this kind of market.
Before we arrive at a mismatch between AFP supply and demand, alternative sources of antifreeze production must be investigated and developed. Recombinant AFP production that uses fermentation technologies, mammalian cell lines, transgenic plants, and transgenic dairy animals (92 ) are currently being explored as means of meeting this future demand.
Acknowlegments
The authors thank A/F Protein and Michael Erisman for providing information about some of the market potentials.
This research was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC), the Industrial Research Assistance Program-National Research Council (IRAP-NRC), the Atlantic Canada Opportunities Agency (ACOA), and A/F Protein Canada Inc.
For more information
A/F Protein Canada Inc. is a development-stage biotechnology company wholly owned by A/F Protein Inc. (USA), with operations in Newfoundland and Prince Edward Island. Its mission is to develop the commercial potential of two distinct products: AFPs and, through its Aqua Bounty Farms division, AquAdvantage fish, which are genetically modified for rapid growth. Find out more about the company at its Web site, http://www.afprotein.com.
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