TRANSGENIC FISH FOR AQUACULTURE
Garth L. Fletcherl and Peter L. Davies2 IOcean Sciences Centre Memorial University of Newfoundland St. John's, Newfoundland, Canada AIC 5S7 2Department of Biochemistry Queen's University Kingston, Ontario, Canada K7L 3N6 INTRODUCTION This review focuses on the technology currently being applied to produce transgenic fish, fish containing novel gene constructs that were experimentally introduced into their genome. A number of excellent overviews on this subject have been published in recent years (1-5). Thus rather than repeat what has already been well stated we have, where possible, attempted to examine critically the published results in order to determine the factors that have or have not been established as being important to the efficient production of stable lines of transgenic fish. We hope that this exercise will be useful to researchers currently applying, or contemplating applying, transgenic technology to fish, and that it will assist all of us in the design of experiments that will enable the production of transgenic fish to be as successful as the production of transgenic mice. The driving force behind the application of transgenic technology to fish is the desire to produce genetically superior broodstocks for food production. As with all fields of applied biology the success of this technology will be judged solely by the ultimate product. Thus in order to be successful the scientific community must work closely with the aquaculture industry fully to understand its problems, both from a production and marketing point of view. There is little point in promoting the value of the technology if the product can never be marketed. We came into the field of transgenics some eight years ago in response to a very practical problem facing the aquaculture industry. During the winter the marine environment along most of the Atlantic coast of Canada is characterized by subzero (0 to -1.8°C) water temperatures and the frequent occurrence of surface ice. Salmonids, and most other commercially important fish, freeze if they come into contact with ice at temperatures below -.07°C (6). Therefore sea-cage Genetic Engineering. Vol. /3 Edited by J.K. Setlow, Plenum Press, New York, 1991
331
332
G. J. FLETCHER AND P. L. DAVIES
culture is almost entirely restricted to relatively small areas at the southern edge of eastern Canada where the occurrence of freezing conditions is rare (7,8). Since we had been studying the basis of freezing resistance in fish for many years it seemed reasonable and desirable to respond to the needs of a fledgling industry (9). Since we had already isolated antifreeze protein genes from a freeze-resistant fish, could we transfer them to Atlantic salmon? Can we produce freeze-resistant fish (salmon, trout, charr, halibut, etc.) and thus facilitate the development of aquaculture in northern regions where the only limiting factor is the winter freezing temperatures? In Newfoundland the development of aquaculture enterprises could have a significant impact on the economic viability of many communities scattered along most of the coastline. Progress in the application of transgenic technology to fish aquaculture has been very rapid, with outdoor performance tests of rapidly growing transgenic carp (Cypdnus carpio), containing a rainbow trout growth hormone gene construct, being carried out within four years of the first published reports on the successful production of transgenic fish with cloned genes (10-13). The reason for this advanced stage of development of transgenic fish compared with transgenic farm animals is largely attributable to the relative ease with which such research can be carried out on fish. Fish, in contrast to mammals, produce large numbers of eggs (hundreds to thousands) (Table 2) and in many species, particularly salmonids, these eggs can be readily obtained during the reproductive season by gently squeezing (stripping) them out of the genital pore. In addition the eggs of most species under study are very robust and relatively large, ranging from I mm to 7 mm in diameter (Table 2). Their large size and general robustness simplifies handling and microinjection procedures, all of which can be carried out without the necessity of sterile conditions. Since fertilization of the eggs is external it can be delayed for a considerable period of time following egg collection. For example, salmonid eggs and sperm can be transported large distances and fertilization can be delayed for days without significant changes in their viability. Cultivation of the fertilized eggs is, in most cases, very easy, all that is required is an adequate water supply. WHAT CAN TRANSGENICS DO FOR FISH CULTURE? It is evident that the potential economic benefits of transgenic technology to aquaculture are paramount. The isolation and construction of genes responsible for desirable traits, and their transfer to broodstocks, could provide a quantum leap over traditional selection and breeding methods. In addition, new traits not present in a genome can be transferred to it from unrelated species, enabling the production of new phenotypes. There are many changes in the genetic make-up of fish that have been identified as desirable (Table 1). However, for the present, most of these changes can only be regarded as items on a "wish" list, because we are many years from identifying the responsible genes. At the present time the only available genes that have a high potential value to aquaculture are the growth hormone (GH) and fish antifreeze protein (AFP) genes. The growth-promoting effects of parenterally administered mammalian and piscine GH on fish have been well documented (14-25). Similarly, intraperitoneal injections of purified winter flounder AFP have been
TRANSGENIC FISH FOR AQUACULTURE
333
Table 1 What Can Transgenic Technology do for Fish Improve economics of fish culture - increase growth rates - increase overall size - increase dress-out percentage - improve feed conversion efficiencies - utilize low cost diets (carbohydrates as opposed to protein) - improve cold tolerance - improve freeze resistance - improve disease resistance - increase brood stock fecundity - control smolting and reproduction - reduce aggression 2
Tailor fish for the market - external appearance; food fish or exotic tropicals - flesh color, flavor, texture - fatty acid composition
3
Fish as bioreactors - production of medically important compounds - production of commercially useful non-medical compounds
4
Basic research aimed at understanding developmental, growth and reproductive processes in fish.
shown to increase the freezing resistance of rainbow trout (26). In addition these AFP can also improve the cold hardiness of plant tissues (27). Since the administration of GH and AFP can produce the desired phenotypic effects in fish it remains to be determined whether the genes coding for these proteins can be stably integrated into the genome and expressed at levels appropriate to their function. In terms of expression, the antifreeze and growth hormone genes present two very different problems to the investigator. In order to be effective the antifreeze genes must express large quantities of protein that are secreted into the extracellular space to build up levels of 5 to 20 mg/ml (28). Since antifreeze genes are normally expressed in the liver of fish possessing them it is theoretically appropriate to transfer the entire structural gene, including the regulating sequences, and obtain adequate expression. However antifreeze genes are usually present as large multigene families containing up to 150 copies and in addition there is a strong correlation between gene copy number and the level of antifreeze protein expressed in natural populations of fish (95,96). Thus, if only a few gene copies are integrated into the transgenic fish the level of expression may not be sufficient to improve its freeze resistance. The low levels of expression that we have observed to date in our salmon, transgenic for winter flounder AFP gene,
334
G. J. FLETCHER AND P. L. DAVIES
suggest that this may indeed be the case (29). The solution to this problem may be in identifying a sufficiently strong promoter/enhancer, or engineering a protein
that is resistant to normal enzymatic degradation. In contrast to antifreeze proteins, blood growth hormone levels in fish are very low, typically less than 50 nglml. Therefore it may not be necessary for the gene to have a strong promoter. However, it is necessary to modify its tissuespecific expression, because there is little value in transferring GH genes if they can only be expressed in the pituitary gland under homeostatic control via the hypothalamus. In order to avoid this problem investigators construct chimeric GH genes using promoters that would be expected to be expressed in tissues other than the pituitary gland. At the present time promoters include mouse MT-l, RSV and SV40. In our own research we have coupled the ocean pout antifreeze promoter to the chinook salmon growth hormone (30). A number of groups have reported increased growth rates in fish transgenic for GH (31-33). However the research is still in its infancy, thus it is too early fully to assess the promise and problems posed by the use of the various GH gene constructs. The potential advantages of transgenic technology for fish research and production can be grouped into the four areas outlined in Table 1. Most current research in the field can be considered under the umbrella of improving the economics of fish culture, a highly justifiable target that has provided the reason for much of the research to be funded. However, although commercial aquaculture is providing the focus for transgenic technology, the research carried out to accomplish this end will generate a basic understanding of gene regulation, developmental biology, physiology and biochemistry of fish in general. This is patently evident in our own research fields, where funds provided by the Strategic Grant Program of the Natural Sciences and Engineering Research Council of Canada to produce freeze-resistant salmon for aquaculture has provided us with the means to examine closely the antifreeze genes and their regulation in the fish that normally possess them (34,35). What has become evident to all of us is that the successful application of transgenic technology to the production of commercially valuable fish requires the cooperative efforts of molecular and developmental biologists, biochemists, physiologists, geneticists, fish biologists and aquaculture specialists. This bringing together of expertise from all fields of biology to focus primarily on fish as food cannot help but supply the critical mass necessary for the establishment of fish as model vertebrates so eloquently argued for by Dennis Powers (36) and Marcia Barinaga (37). FISH SPECIES UNDER STUDY A few characteristics of most of the fish species currently being used in transgenic studies are presented in Table 2. The information contained therein demonstrates the diversity of the species under study, and provides information pertinent to understanding some of the experimental problems posed by their use in transgenic research. As a result of its applied target, most research on transgenic fish is being carried out on representatives of the world's most important culture species. In an overview of global aquaculture production in 1987, Nash and Kensler (38) present statistics indicating that the cyprinids (carps) (3,939,616 metric tons (t), salmonids
Age
30-40
11-14 (88) 3 2.5-5
2-6 yr
3-6 mo (88) 3 mo 2-3 mo -100 g (56)
-5 kg
40-50
2-3 yr once/year once/year year round year round year round
once/year
once/year
> 18 (87)
2-5 yr (87) 0.9-4.5 kg (88)
once/year
>5
> 50,000 5,000-10,000 70-300 (88) 20-40 150-400 (92)
7,000-30,000 (88) 10,000-30,000
> 2,000 (87)
> 100,000
800-1,000
5,000-12,000
No. at Spawning
Eggs
2.5 (56) 1.0 1.0
-1.3
1.5-2.0
2.3
1.2-1.5
1.5 (85)
1-1.5
3-5
5-7
Size (mm)
7 21-22 27-30 27 26
7-8
26-27
20-25
20
10
8
Temperature (0C)
4hr 30-60 min 30 min (56) Ihr 35 min
30-50 min (87) 90 min (46)
30 min (32)
25 5 4 (55) 10 4
10-12
5-10 (88)
4 (86)
3-4
-35 (85)
75-80
13-15 hr 6-8 hr
Hatch (Days)
1st Cleavage
Time to
Atlantic salmon (Salmo saJUj; Rainbow trout (Oncorhynchus mykiss, formally Salmo gairdnen); Common carp (Cyprinus carpio); Goldfish (Carrasium auratus); Channel catfish (lctalums punctatus); Northern pike (Esox Jucius); Walleye (Stizostedion V vitreum); Loach (Misgumus fossilis); Tilapia (Oreochromis nilaticus); Medaka (Oryzias Jatipes); Zebrafish (Brachydanio rerio). References are numbers in parentheses.
Channel catfish Northern pike (89) Walleye (90) Loach (47) Tilapia Medaka (2) Zebrafish (91)
once/year
once/year
Spawning Frequency
25-40
3-7 kg
Weight
once/year
Length (cm)
15-25
Atlantic 3-4 yr salmon (83) Rainbow trout 1-5 yr (83) Common carp 2-4 yr (84) Goldfish 1+ yr
Species
Sexual Maturity
Table 2 Fish Currently Used for Transgenic Studies
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336
G. J. FLETCHER AND P. L. DAVIES
(salmon and trout) (321,376 t), cichlids (til apia) (246,399 t) and catfish (169,982 t) make up 80% of world production of cultured freshwater fish. The species named in Table 2 approximate 25% of world production. Apart from tilapia, most of the commercial species are relatively large and have long generation times. Thus they are ill-suited as models to study transgenism. This role appears better suited for tropical species such as the medaka and zebrafish, which are small, relatively easy to culture and have short generation times. One drawback of these small species is the difficulty in obtaining blood samples without killing the fish. At the present time most fish blood growth hormone assays require relatively large plasma samples (50 to 100 /-11). Tilapia species, although larger than the medaka and zebrafish, also have short generation times. Therefore they have potential as model fish and thus provide the researcher with the unique opportunity of coupling basic and applied research on the same species. GENE TRANSFER TECHNIQUES To date most of the gene transfer experiments on fish have capitalized on the success of the microinjection techniques developed for mammals. However since fish eggs differ in many respects from those of mammals various details of the injection techniques had to be modified accordingly. In the ensuing sections an attempt is made to evaluate critically various aspects of the microinjection procedures currently being used for fish. In order to do this, techniques to penetrate the chorion, constituents of the injection buffer, injection target and timing relative to fertilization, DNA form and concentration are evaluated somewhat independently of one another. However it is accepted that the successful production of stable lines of transgenic fish in the most efficient way will depend upon an optimum combination of these factors. Fish Egg Structure The large size of most teleost fish eggs is due primarily to their quantity of yolk (Figure 1). Upon ovulation and release from the body cavity (spawning) the egg is encased in a thick proteinaceous outer membrane known as the chorion or zona radiata (30 to 40 /-1 thick, salmonids) that serves to protect the egg. This membrane is impermeable to sperm. Therefore all teleost fish eggs that are fertilized following spawning have a single opening (micropyle) in the chorion just large enough to allow the entry of a single spermatozoa. Immediately underlying the chorion, and closely adhering to it in the unfertilized egg, is the vitelline (plasma) membrane. The central part of the egg is filled with yolk while the cytoplasm is present as a thin (~ 40 /-1) peripheral layer that is somewhat thicker (~ 100 /-1) at the animal pole (39). Meiosis in fishes, as in most chordates, is blocked at the metaphase stage of the second maturation division. The metaphase II plate and the first polar body can be found at the animal pole in close proximity to the micropyle in unfertilized eggs (39,40) (Figure 1). Immediately upon fertilization the egg is activated by contact between the fertilizing sperm and the ooplasm. This activation results in the completion of meiosis and the extrusion of the second polar body under the chorion. At the same time the cortical alveoli, located in the peripheral cytoplasm,
337
TRANSGENIC FISH FOR AOUACUL TURE
BLASTODISC
A. UHFERTILIZED
UITELLIHE HEHBRAHE
B. FERTILIZED
Figure 1. Teleost egg. discharge their colloidal contents under the chorion resulting in the formation of the perivitelline space and fluid. These changes are accompanied by migration of the peripheral cytoplasm to concentrate at the animal pole, forming the blastodisc (Figure 1). The formation of the fluid-filled perivitelline space allows the developing egg to rotate freely within the chorion. In most eggs the blastodisc remains on top and there is no longer any link between its location and that of the micropyle. In many species the chorion itself becomes a very hard protective shell (39).
Egg Injection It has been generally assumed that the most effective place to inject gene constructs is directly into the pronucleus or nucleus of the egg before the onset of cleavage. The rationale behind this is that integration into the genome prior to first cleavage will result in all of the organism's cells containing the same copy number of the foreign gene integrated into the same chromosomal sites. Although it is evident that this result can be achieved in mammals (41), available evidence from studies on fish is not as encouraging. The production of mosaics, indicating integration at or after the two-cell stage, appears to be the rule rather than the exception. One of the difficulties encountered with fish eggs lies with the inability of most investigators to visualize the nucleus or pronucleus using conventional light microscopy. In most cases, if not all, this is attributable to the opaqueness of the chorion and/or the cytoplasm. Rokkones et al. (42) reported unsuccessful attempts to increase the visibility of the pronucleus in salmonid eggs by centrifugation or by vital staining using fluorescent dyes. Centrifugation has proved effective with pig eggs (43). Yamaha et al. (44) have successfully located the female pronucleus in dechorinated goldfish eggs using the fluorescent dye, Hoechst 33342. However no studies have been carried out to determine the effects of this dye, or the ultraviolet light necessary for its fluorescence, on the viability of the eggs. Hammer et al. (43) have used this technique on mammals and report that the procedure damages the ovum.
338
G. J. FLETCHER AND P. L. DAVIES
Since, in most instances, nuclear structures cannot be located, most investigators have opted for cytoplasmic injections following fertilization. Cytoplasmic injections, although not as effective as nuclear injections, have been successful in mice (45). The target for cytoplasmic injections in fish eggs is the thin layer of ooplasm just under the chorion or the developing blastodisc (Figure 1). If the needle penetrates the yolk, the eggs usually die. In the following sections, the various injection strategies and procedures are described, and their success evaluated on the basis of survival of injected eggs, integration of the exogenous gene into the host DNA, and the degree of mosaicism observed. Nuclear Injections Ozato et al. (13) appear to be the only group that have successfully overcome the problem of inserting gene constructs into the nucleus. In order to do this they capitalized on their knowledge of gonadal development in the medaka and surgically removed the oocytes from the ovary prior to ovulation. At the time of removal the cytoplasm is transparent and the large nucleus (germinal vesicle) (120 to 150 Ilm), which is in the prophase of the second meiotic division, is clearly visible and can be readily injected. Following injection, the oocytes are cultured until maturation, fertilized and allowed to develop. This technique appears to be reasonably successful in that 50% of the injected eggs developed into normal embryos, of which 50% contained the injected sequence (13). However these authors found, using DNA-DNA in situ hybridization techniques, that the nuclei containing the exogenous sequence were distributed mosaicially in every tissue. Thus it appears that injecting fish egg pronuclei does not guarantee integration into all cells. It is likely that procedures similar to those of Ozato et al. (13) can be developed for other species of fish. However at the present time the additional effort does not appear to be justified, at least for commercial species, because the results obtained to date do not differ appreciably from those reported by others using less sophisticated techniques. Injection Through Soft Chorions In fish such as the common carp, channel catfish and loach, the chorion remains relatively soft following fertilization and can be penetrated readily with glass needles (2 to 10 Ilm, carp and catfish, 20 to 35 Ilm, loach) (32,46,47). Dunham et al. (46), and Zhang et al. (32) used as the target for injection the blastodisc prior to cleavage, whereas Korzh (47) injects the yolky interphase immediately adjacent to the blastodisc. In the catfish, Dunham et al. (46) report an average mortality rate of 87% prior to hatching, compared with 90% (hatch) 74% (0.5-50) (6 mo) yes no
hGH SV40 linear (yes) 3.95 25 Ix10 8 (500)
29,8,12,20%/-
no
60-80%/>90% (hatch) 38% (2-40) (6 mo)
rGH mouse MT linear (yes) 6.6 10 3x10 7 (200)
10J.l.m 20 nl
cut hole blastodisc ---->
10
16%/-
60-80%/>90% (hatch) 40% (2-40) (1 yr) yes no
10 - (200)
hGH mouse MT linear (yes)
cut hole cut hole blastodisc blastodisc ----> 1 cell 3-5 hr after fertilization 10llm 10J.l.m 10 J.l.m 20 nl 20 nl 20 nl mM Tris HCI 50 mM NaCl 1 mM EDT A -------->
cut hole blastodisc ---->
10
hGH SV40 circular (yes)
cut hole blastodisc I cell 2-6 hr after fertilization 10 J.l.m 20 nl I cell> 2 hr after fertilization 10 J.l.m 10 J.l.m 10 nl 10 nl 10 mM Tris HCI 1 mM EDTA, pH 8.0
5-10
Inheritance F /F 2 (%) Expression F/F2 (%) (42) Reference
DNA Construct Promoter Form (vector) Length (kb) Conc. (J.l.glml) Inject copy no. (pg) % Survival inj/cont (age) "Integration" % (Copy No.) Mosaic F0 expression (%)
Temperature (0C) Chorion solution Site injected Development stage Needle diameter Volume injected Buffer
Table 3 (continued) Transgenic Fish Swnmary Rainbow Trout
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Garth L. Fletcherl and Peter L. Davies2 IOcean Sciences Centre Memorial University of Newfoundland St. John's, Newfoundland, Canada AIC 5S7 2Department of Biochemistry Queen's University Kingston, Ontario, Canada K7L 3N6 INTRODUCTION This review focuses on the technology currently being applied to produce transgenic fish, fish containing novel gene constructs that were experimentally introduced into their genome. A number of excellent overviews on this subject have been published in recent years (1-5). Thus rather than repeat what has already been well stated we have, where possible, attempted to examine critically the published results in order to determine the factors that have or have not been established as being important to the efficient production of stable lines of transgenic fish. We hope that this exercise will be useful to researchers currently applying, or contemplating applying, transgenic technology to fish, and that it will assist all of us in the design of experiments that will enable the production of transgenic fish to be as successful as the production of transgenic mice. The driving force behind the application of transgenic technology to fish is the desire to produce genetically superior broodstocks for food production. As with all fields of applied biology the success of this technology will be judged solely by the ultimate product. Thus in order to be successful the scientific community must work closely with the aquaculture industry fully to understand its problems, both from a production and marketing point of view. There is little point in promoting the value of the technology if the product can never be marketed. We came into the field of transgenics some eight years ago in response to a very practical problem facing the aquaculture industry. During the winter the marine environment along most of the Atlantic coast of Canada is characterized by subzero (0 to -1.8°C) water temperatures and the frequent occurrence of surface ice. Salmonids, and most other commercially important fish, freeze if they come into contact with ice at temperatures below -.07°C (6). Therefore sea-cage Genetic Engineering. Vol. /3 Edited by J.K. Setlow, Plenum Press, New York, 1991
331
332
G. J. FLETCHER AND P. L. DAVIES
culture is almost entirely restricted to relatively small areas at the southern edge of eastern Canada where the occurrence of freezing conditions is rare (7,8). Since we had been studying the basis of freezing resistance in fish for many years it seemed reasonable and desirable to respond to the needs of a fledgling industry (9). Since we had already isolated antifreeze protein genes from a freeze-resistant fish, could we transfer them to Atlantic salmon? Can we produce freeze-resistant fish (salmon, trout, charr, halibut, etc.) and thus facilitate the development of aquaculture in northern regions where the only limiting factor is the winter freezing temperatures? In Newfoundland the development of aquaculture enterprises could have a significant impact on the economic viability of many communities scattered along most of the coastline. Progress in the application of transgenic technology to fish aquaculture has been very rapid, with outdoor performance tests of rapidly growing transgenic carp (Cypdnus carpio), containing a rainbow trout growth hormone gene construct, being carried out within four years of the first published reports on the successful production of transgenic fish with cloned genes (10-13). The reason for this advanced stage of development of transgenic fish compared with transgenic farm animals is largely attributable to the relative ease with which such research can be carried out on fish. Fish, in contrast to mammals, produce large numbers of eggs (hundreds to thousands) (Table 2) and in many species, particularly salmonids, these eggs can be readily obtained during the reproductive season by gently squeezing (stripping) them out of the genital pore. In addition the eggs of most species under study are very robust and relatively large, ranging from I mm to 7 mm in diameter (Table 2). Their large size and general robustness simplifies handling and microinjection procedures, all of which can be carried out without the necessity of sterile conditions. Since fertilization of the eggs is external it can be delayed for a considerable period of time following egg collection. For example, salmonid eggs and sperm can be transported large distances and fertilization can be delayed for days without significant changes in their viability. Cultivation of the fertilized eggs is, in most cases, very easy, all that is required is an adequate water supply. WHAT CAN TRANSGENICS DO FOR FISH CULTURE? It is evident that the potential economic benefits of transgenic technology to aquaculture are paramount. The isolation and construction of genes responsible for desirable traits, and their transfer to broodstocks, could provide a quantum leap over traditional selection and breeding methods. In addition, new traits not present in a genome can be transferred to it from unrelated species, enabling the production of new phenotypes. There are many changes in the genetic make-up of fish that have been identified as desirable (Table 1). However, for the present, most of these changes can only be regarded as items on a "wish" list, because we are many years from identifying the responsible genes. At the present time the only available genes that have a high potential value to aquaculture are the growth hormone (GH) and fish antifreeze protein (AFP) genes. The growth-promoting effects of parenterally administered mammalian and piscine GH on fish have been well documented (14-25). Similarly, intraperitoneal injections of purified winter flounder AFP have been
TRANSGENIC FISH FOR AQUACULTURE
333
Table 1 What Can Transgenic Technology do for Fish Improve economics of fish culture - increase growth rates - increase overall size - increase dress-out percentage - improve feed conversion efficiencies - utilize low cost diets (carbohydrates as opposed to protein) - improve cold tolerance - improve freeze resistance - improve disease resistance - increase brood stock fecundity - control smolting and reproduction - reduce aggression 2
Tailor fish for the market - external appearance; food fish or exotic tropicals - flesh color, flavor, texture - fatty acid composition
3
Fish as bioreactors - production of medically important compounds - production of commercially useful non-medical compounds
4
Basic research aimed at understanding developmental, growth and reproductive processes in fish.
shown to increase the freezing resistance of rainbow trout (26). In addition these AFP can also improve the cold hardiness of plant tissues (27). Since the administration of GH and AFP can produce the desired phenotypic effects in fish it remains to be determined whether the genes coding for these proteins can be stably integrated into the genome and expressed at levels appropriate to their function. In terms of expression, the antifreeze and growth hormone genes present two very different problems to the investigator. In order to be effective the antifreeze genes must express large quantities of protein that are secreted into the extracellular space to build up levels of 5 to 20 mg/ml (28). Since antifreeze genes are normally expressed in the liver of fish possessing them it is theoretically appropriate to transfer the entire structural gene, including the regulating sequences, and obtain adequate expression. However antifreeze genes are usually present as large multigene families containing up to 150 copies and in addition there is a strong correlation between gene copy number and the level of antifreeze protein expressed in natural populations of fish (95,96). Thus, if only a few gene copies are integrated into the transgenic fish the level of expression may not be sufficient to improve its freeze resistance. The low levels of expression that we have observed to date in our salmon, transgenic for winter flounder AFP gene,
334
G. J. FLETCHER AND P. L. DAVIES
suggest that this may indeed be the case (29). The solution to this problem may be in identifying a sufficiently strong promoter/enhancer, or engineering a protein
that is resistant to normal enzymatic degradation. In contrast to antifreeze proteins, blood growth hormone levels in fish are very low, typically less than 50 nglml. Therefore it may not be necessary for the gene to have a strong promoter. However, it is necessary to modify its tissuespecific expression, because there is little value in transferring GH genes if they can only be expressed in the pituitary gland under homeostatic control via the hypothalamus. In order to avoid this problem investigators construct chimeric GH genes using promoters that would be expected to be expressed in tissues other than the pituitary gland. At the present time promoters include mouse MT-l, RSV and SV40. In our own research we have coupled the ocean pout antifreeze promoter to the chinook salmon growth hormone (30). A number of groups have reported increased growth rates in fish transgenic for GH (31-33). However the research is still in its infancy, thus it is too early fully to assess the promise and problems posed by the use of the various GH gene constructs. The potential advantages of transgenic technology for fish research and production can be grouped into the four areas outlined in Table 1. Most current research in the field can be considered under the umbrella of improving the economics of fish culture, a highly justifiable target that has provided the reason for much of the research to be funded. However, although commercial aquaculture is providing the focus for transgenic technology, the research carried out to accomplish this end will generate a basic understanding of gene regulation, developmental biology, physiology and biochemistry of fish in general. This is patently evident in our own research fields, where funds provided by the Strategic Grant Program of the Natural Sciences and Engineering Research Council of Canada to produce freeze-resistant salmon for aquaculture has provided us with the means to examine closely the antifreeze genes and their regulation in the fish that normally possess them (34,35). What has become evident to all of us is that the successful application of transgenic technology to the production of commercially valuable fish requires the cooperative efforts of molecular and developmental biologists, biochemists, physiologists, geneticists, fish biologists and aquaculture specialists. This bringing together of expertise from all fields of biology to focus primarily on fish as food cannot help but supply the critical mass necessary for the establishment of fish as model vertebrates so eloquently argued for by Dennis Powers (36) and Marcia Barinaga (37). FISH SPECIES UNDER STUDY A few characteristics of most of the fish species currently being used in transgenic studies are presented in Table 2. The information contained therein demonstrates the diversity of the species under study, and provides information pertinent to understanding some of the experimental problems posed by their use in transgenic research. As a result of its applied target, most research on transgenic fish is being carried out on representatives of the world's most important culture species. In an overview of global aquaculture production in 1987, Nash and Kensler (38) present statistics indicating that the cyprinids (carps) (3,939,616 metric tons (t), salmonids
Age
30-40
11-14 (88) 3 2.5-5
2-6 yr
3-6 mo (88) 3 mo 2-3 mo -100 g (56)
-5 kg
40-50
2-3 yr once/year once/year year round year round year round
once/year
once/year
> 18 (87)
2-5 yr (87) 0.9-4.5 kg (88)
once/year
>5
> 50,000 5,000-10,000 70-300 (88) 20-40 150-400 (92)
7,000-30,000 (88) 10,000-30,000
> 2,000 (87)
> 100,000
800-1,000
5,000-12,000
No. at Spawning
Eggs
2.5 (56) 1.0 1.0
-1.3
1.5-2.0
2.3
1.2-1.5
1.5 (85)
1-1.5
3-5
5-7
Size (mm)
7 21-22 27-30 27 26
7-8
26-27
20-25
20
10
8
Temperature (0C)
4hr 30-60 min 30 min (56) Ihr 35 min
30-50 min (87) 90 min (46)
30 min (32)
25 5 4 (55) 10 4
10-12
5-10 (88)
4 (86)
3-4
-35 (85)
75-80
13-15 hr 6-8 hr
Hatch (Days)
1st Cleavage
Time to
Atlantic salmon (Salmo saJUj; Rainbow trout (Oncorhynchus mykiss, formally Salmo gairdnen); Common carp (Cyprinus carpio); Goldfish (Carrasium auratus); Channel catfish (lctalums punctatus); Northern pike (Esox Jucius); Walleye (Stizostedion V vitreum); Loach (Misgumus fossilis); Tilapia (Oreochromis nilaticus); Medaka (Oryzias Jatipes); Zebrafish (Brachydanio rerio). References are numbers in parentheses.
Channel catfish Northern pike (89) Walleye (90) Loach (47) Tilapia Medaka (2) Zebrafish (91)
once/year
once/year
Spawning Frequency
25-40
3-7 kg
Weight
once/year
Length (cm)
15-25
Atlantic 3-4 yr salmon (83) Rainbow trout 1-5 yr (83) Common carp 2-4 yr (84) Goldfish 1+ yr
Species
Sexual Maturity
Table 2 Fish Currently Used for Transgenic Studies
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336
G. J. FLETCHER AND P. L. DAVIES
(salmon and trout) (321,376 t), cichlids (til apia) (246,399 t) and catfish (169,982 t) make up 80% of world production of cultured freshwater fish. The species named in Table 2 approximate 25% of world production. Apart from tilapia, most of the commercial species are relatively large and have long generation times. Thus they are ill-suited as models to study transgenism. This role appears better suited for tropical species such as the medaka and zebrafish, which are small, relatively easy to culture and have short generation times. One drawback of these small species is the difficulty in obtaining blood samples without killing the fish. At the present time most fish blood growth hormone assays require relatively large plasma samples (50 to 100 /-11). Tilapia species, although larger than the medaka and zebrafish, also have short generation times. Therefore they have potential as model fish and thus provide the researcher with the unique opportunity of coupling basic and applied research on the same species. GENE TRANSFER TECHNIQUES To date most of the gene transfer experiments on fish have capitalized on the success of the microinjection techniques developed for mammals. However since fish eggs differ in many respects from those of mammals various details of the injection techniques had to be modified accordingly. In the ensuing sections an attempt is made to evaluate critically various aspects of the microinjection procedures currently being used for fish. In order to do this, techniques to penetrate the chorion, constituents of the injection buffer, injection target and timing relative to fertilization, DNA form and concentration are evaluated somewhat independently of one another. However it is accepted that the successful production of stable lines of transgenic fish in the most efficient way will depend upon an optimum combination of these factors. Fish Egg Structure The large size of most teleost fish eggs is due primarily to their quantity of yolk (Figure 1). Upon ovulation and release from the body cavity (spawning) the egg is encased in a thick proteinaceous outer membrane known as the chorion or zona radiata (30 to 40 /-1 thick, salmonids) that serves to protect the egg. This membrane is impermeable to sperm. Therefore all teleost fish eggs that are fertilized following spawning have a single opening (micropyle) in the chorion just large enough to allow the entry of a single spermatozoa. Immediately underlying the chorion, and closely adhering to it in the unfertilized egg, is the vitelline (plasma) membrane. The central part of the egg is filled with yolk while the cytoplasm is present as a thin (~ 40 /-1) peripheral layer that is somewhat thicker (~ 100 /-1) at the animal pole (39). Meiosis in fishes, as in most chordates, is blocked at the metaphase stage of the second maturation division. The metaphase II plate and the first polar body can be found at the animal pole in close proximity to the micropyle in unfertilized eggs (39,40) (Figure 1). Immediately upon fertilization the egg is activated by contact between the fertilizing sperm and the ooplasm. This activation results in the completion of meiosis and the extrusion of the second polar body under the chorion. At the same time the cortical alveoli, located in the peripheral cytoplasm,
337
TRANSGENIC FISH FOR AOUACUL TURE
BLASTODISC
A. UHFERTILIZED
UITELLIHE HEHBRAHE
B. FERTILIZED
Figure 1. Teleost egg. discharge their colloidal contents under the chorion resulting in the formation of the perivitelline space and fluid. These changes are accompanied by migration of the peripheral cytoplasm to concentrate at the animal pole, forming the blastodisc (Figure 1). The formation of the fluid-filled perivitelline space allows the developing egg to rotate freely within the chorion. In most eggs the blastodisc remains on top and there is no longer any link between its location and that of the micropyle. In many species the chorion itself becomes a very hard protective shell (39).
Egg Injection It has been generally assumed that the most effective place to inject gene constructs is directly into the pronucleus or nucleus of the egg before the onset of cleavage. The rationale behind this is that integration into the genome prior to first cleavage will result in all of the organism's cells containing the same copy number of the foreign gene integrated into the same chromosomal sites. Although it is evident that this result can be achieved in mammals (41), available evidence from studies on fish is not as encouraging. The production of mosaics, indicating integration at or after the two-cell stage, appears to be the rule rather than the exception. One of the difficulties encountered with fish eggs lies with the inability of most investigators to visualize the nucleus or pronucleus using conventional light microscopy. In most cases, if not all, this is attributable to the opaqueness of the chorion and/or the cytoplasm. Rokkones et al. (42) reported unsuccessful attempts to increase the visibility of the pronucleus in salmonid eggs by centrifugation or by vital staining using fluorescent dyes. Centrifugation has proved effective with pig eggs (43). Yamaha et al. (44) have successfully located the female pronucleus in dechorinated goldfish eggs using the fluorescent dye, Hoechst 33342. However no studies have been carried out to determine the effects of this dye, or the ultraviolet light necessary for its fluorescence, on the viability of the eggs. Hammer et al. (43) have used this technique on mammals and report that the procedure damages the ovum.
338
G. J. FLETCHER AND P. L. DAVIES
Since, in most instances, nuclear structures cannot be located, most investigators have opted for cytoplasmic injections following fertilization. Cytoplasmic injections, although not as effective as nuclear injections, have been successful in mice (45). The target for cytoplasmic injections in fish eggs is the thin layer of ooplasm just under the chorion or the developing blastodisc (Figure 1). If the needle penetrates the yolk, the eggs usually die. In the following sections, the various injection strategies and procedures are described, and their success evaluated on the basis of survival of injected eggs, integration of the exogenous gene into the host DNA, and the degree of mosaicism observed. Nuclear Injections Ozato et al. (13) appear to be the only group that have successfully overcome the problem of inserting gene constructs into the nucleus. In order to do this they capitalized on their knowledge of gonadal development in the medaka and surgically removed the oocytes from the ovary prior to ovulation. At the time of removal the cytoplasm is transparent and the large nucleus (germinal vesicle) (120 to 150 Ilm), which is in the prophase of the second meiotic division, is clearly visible and can be readily injected. Following injection, the oocytes are cultured until maturation, fertilized and allowed to develop. This technique appears to be reasonably successful in that 50% of the injected eggs developed into normal embryos, of which 50% contained the injected sequence (13). However these authors found, using DNA-DNA in situ hybridization techniques, that the nuclei containing the exogenous sequence were distributed mosaicially in every tissue. Thus it appears that injecting fish egg pronuclei does not guarantee integration into all cells. It is likely that procedures similar to those of Ozato et al. (13) can be developed for other species of fish. However at the present time the additional effort does not appear to be justified, at least for commercial species, because the results obtained to date do not differ appreciably from those reported by others using less sophisticated techniques. Injection Through Soft Chorions In fish such as the common carp, channel catfish and loach, the chorion remains relatively soft following fertilization and can be penetrated readily with glass needles (2 to 10 Ilm, carp and catfish, 20 to 35 Ilm, loach) (32,46,47). Dunham et al. (46), and Zhang et al. (32) used as the target for injection the blastodisc prior to cleavage, whereas Korzh (47) injects the yolky interphase immediately adjacent to the blastodisc. In the catfish, Dunham et al. (46) report an average mortality rate of 87% prior to hatching, compared with 90% (hatch) 74% (0.5-50) (6 mo) yes no
hGH SV40 linear (yes) 3.95 25 Ix10 8 (500)
29,8,12,20%/-
no
60-80%/>90% (hatch) 38% (2-40) (6 mo)
rGH mouse MT linear (yes) 6.6 10 3x10 7 (200)
10J.l.m 20 nl
cut hole blastodisc ---->
10
16%/-
60-80%/>90% (hatch) 40% (2-40) (1 yr) yes no
10 - (200)
hGH mouse MT linear (yes)
cut hole cut hole blastodisc blastodisc ----> 1 cell 3-5 hr after fertilization 10llm 10J.l.m 10 J.l.m 20 nl 20 nl 20 nl mM Tris HCI 50 mM NaCl 1 mM EDT A -------->
cut hole blastodisc ---->
10
hGH SV40 circular (yes)
cut hole blastodisc I cell 2-6 hr after fertilization 10 J.l.m 20 nl I cell> 2 hr after fertilization 10 J.l.m 10 J.l.m 10 nl 10 nl 10 mM Tris HCI 1 mM EDTA, pH 8.0
5-10
Inheritance F /F 2 (%) Expression F/F2 (%) (42) Reference
DNA Construct Promoter Form (vector) Length (kb) Conc. (J.l.glml) Inject copy no. (pg) % Survival inj/cont (age) "Integration" % (Copy No.) Mosaic F0 expression (%)
Temperature (0C) Chorion solution Site injected Development stage Needle diameter Volume injected Buffer
Table 3 (continued) Transgenic Fish Swnmary Rainbow Trout
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