The application of gene manipulation to aquaculture

Aquaculture, 85 ( 1990) l-20 Elsevier Science Publishers B.V., Amsterdam

-

Printed

in The Netherlands

The Application of Gene Manipulation to Aquaculture NORMAN

MACLEAN

and DAVID PENMAN*

Department of Biology, Southampton University, Southampton SO9 3TU (Great Britain)

ABSTRACT Maclean, N. and Penman, culture, 85: l-20.

D., 1990. The application

of gene manipulation

to aquaculture.

Aqua-

Both finfish and shellfish are very suitable types of animals to use in terms of the introduction of novel genes into their genomes (transgenic induction). Genetic manipulations involving gynogenesis, androgenesis, triploidy, and sex reversal are already established, and future applications of DNA manipulations may well include population studies by DNA analysis and genetic tagging prior to release. Methods for the production of transgenic fish are considered and success to date with rainbow trout, Atlantic salmon, tilapia. channel catfish, medaka, zebrafish. goldfish, carp and loach is discussed. Transgenic induction techniques normally involve injection of cloned copies of the appropriate gene into the cytoplasm of the fertilized egg by microinjection. Ways of assaying for integration, expression and germ line transmission are reviewed. Candidate genes for transgenic induction in fish include those coding for somatotropin (growth hormone), somatotropin release factor, metallothionein, ‘antifreeze’ proteins, crystallin, esterases, and disease resistance factors (when available); useful promoter sequences include metallothionein, heat shock and those of other tissue-specific genes. The use of genes and promoters from piscine rather than mammalian sources will probably be advantageous both for effective expression and the market image of the product. Transgenic fish are also considered as potential expression systems for pharmaceutical products, and problems of containment and planned release are discussed.

INTRODUCTION

Over the last decade rapid progress has been made in the application of gene manipulation technology to aquaculture. This is not surprising since the farming of both shellfish and fin&h, together with the known biology of the organisms concerned, render the aquaculture situation an excellent one in which to successfully apply this new approach. For most of the species concerned, eggs are freely available in bulk, can be fertilized under controlled conditions, *Present address: Institute of Aquaculture, University of Stirling, StirlingFK9

0044-8486/90/$03.50

0 1990 Elsevier Science Publishers

B.V.

4LA (Great Britain)

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N. MACLEAN AND D. PENMAN

and do not require a return to the female reproductive tract for the completion of development (as is the case with mammals). The availability of cloned gene sequences from many species, including finfish, has suddenly provided the genetic engineer with the necessary material, and especially as specific sequences are now being cloned from finfish gene libraries, the opportunity to manipulate fish with their own native genes will surely obviate the concern about the use of human or mammalian genes. Indeed fish are at present an excellent material for the application of gene technology precisely because the ethical considerations which overshadow widespread genetic manipulation with higher animals, such as mammals, hardly apply to the animals involved in aquaculture. In this review we mainly address the production of transgenic finfish, but since a range of other techniques might also qualify as examples of gene manipulation some passing comment will be made about such methods initially. SOME APPLICATIONS OF GENETIC MANIPULATION TO AQUACULTURE

Gene manipulation is most frequently used exclusively to denote methods which involve manipulation of a gene sequence outside the animal from which the sequence was derived. If the terminology is used in a broader context, however, to cover all situations in which genes are artificially manipulated even without in vitro handling, then some other important applications to aquaculture qualify for comment. 1. Gymgenesis This topic was discussed by Thorgaard (1986). It involves activation of egg development by sperm, without the sperm making a genetic contribution to the resulting embryo. Thus it is a form of entirely maternal inheritance. The technique involves induction of gynogenetic diploidy by induced retention of the second polar body or an enforced block to the first cleavage. 2. Androgenesis This, the complement to gynogenesis, involves production of progeny in which genetic material is derived exclusively from the male, usually by irradiation of eggs prior to fertilization by sperm (Parsons and Thorgaard, 1984)) and diploidization by interference with first cell divisions. 3. Induced polyploidy Polyploid fish which result from traumatic interference with eggs are most frequently triploids, and are produced by treating fertilized eggs with either hydrostatic pressure, temperature shock or chemical treatment. Heat shock is particularly applicable to large batches of eggs, and the technique has found useful application in the production of triploid rainbow trout (Chourrout and Quillet, 1982). Triploids may also be produced by the crossing of diploid indi-

THE APPLICATION OF GENE MANIPC’LATION TO AQUACULTURE

3

viduals with tetraploids (discussed in Thorgaard, 1986). The great attraction of triploid fish is their sterility, and this feature can render them attractive in terms of growth characteristics. More particularly they may be released without fear of contribution to wild-stock gene pools, and this aspect will be discussed later in the context of release of transgenic fish. 4. Sex reversal This is now an established procedure for the production of all-female trout and has been thoroughly discussed by Bye and Lincoln (1986). It involves the treatment of fry with methyltestosterone in food for 700°C days. The female fish are thus induced to become phenotypically male, although still genotypitally female. Semen recovered from such sex-reversed fish (often obtained by surgical removal of testes) is composed of sperm which is entirely female-determining since each sperm carriers only a single X chromosome. Normal eggs fertilized by such sperm yield normal diploid but all-female progeny. 5. Population studies involving gene manipulation Currently most work on fish populations depends on electrophoretic analysis of the protein products of particular gene loci, especially polymorphic loci. The number of suitable loci is normally less than 20 for any species and the method has a number of known shortcomings. Interest had therefore developed in the possible use of DNA rather than protein analysis for determining relationships within populations. A number of laboratories have begun to explore the possible use of mitochondrial DNA analysis to this end (Skibinski, 1985; Edwards and Skibinski, 1987). This approach involves the digestion of mitochondrial DNA with specific restriction enzymes, followed by electrophoretie separation of the resulting DNA fragments. Hybridization to the fragments with a cloned mitochondrial DNA probe, followed by autoradiography, can also be used to improve the resolution of the banding pattern obtained and prevent confusion with the background of nuclear DNA. Prehybridization also reduces the amount of mtDNA necessary for each digest. Other DNA-based approaches involve exploitation of the characteristics of genomic DNA. The chief problem here is that in much of the DNA, changes in base sequence are too frequent to yield useful information about populations or to allow the unequivocal allotment of individuals to their respective subpopulations. Thus the technique of ‘DNA fingerprinting’, which involves the digestion of DNA samples with restriction enzymes followed by hybridization with probe DNA from known genomic minisatellites (hypervariable regions which consist of blocks of short repetition sequences and which are probably non-coding), has found considerable use in establishing unequivocal family relationships in humans (Jeffreys et al., 1985) and other animal species such as birds (Burke and Bruford, 1987 ). However, since only close relatives show similarity, e.g., identical twins show band pattern identity, genetic fingerprint-

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N.MACLEANANDD.PENMAN

ing as presently executed does not lend itself to routine investigation of more distant relationships within or between populations. Another alternative to the study of populations using polymorphic proteins is to compare DNA band patterns of highly repetitious genes such as those coding for ribosomal or transfer RNA. Radio-labelled probes for such sequences could be used to reveal band patterns following DNA digestion with restriction enzymes, much as is done with the DNA fingerprinting methodology, and if suitable probes and restriction enzymes were chosen, it seems likely that such genes, present as hundreds of copies per genome, might offer information at about the correct level of variability. Studies on phylogenetic relationships between individual bacterial and protozoan species have been based on sequence variabilities in the genes for ribosomal RNA (Pace et al., 1986; Sogin et al., 1986). 6. Tagging Production of transgenic fish, by methods discussed later in this review, would permit the development of fish with genetic markers involving trivial and completely inactive gene sequences. Such sequences could be entirely artificial and consist of homopolymers of DNA. If such fish were released and permitted to breed, a predictable proportion of their progeny would also carry the genetic marker, especially if the transgenics were made homozygous for the marker before release; this marker could be readily detected by dot blot hybridization of biopsy material such as blood. Such a genetic tag would permit a form of analysis not feasible with current methods, namely, the introduction of, say, tagged salmon smolts into a river, and later determination of their contribution to future smolt populations in the same river. Of course such a programme would imply the planned release of fish which had been genetically modified, albeit in a trivial way, and it may be that, at least in the short term, objections to such a project might prove insurmountable. If the requirement arose, however, production of such fish would be a relatively routine matter for any laboratory with skills in gene manipulation technology. PRODUCTION OF TRANSGENIC FISH

The discovery of restriction endonuclease enzymes and bacterial plasmids has made possible the cloning of gene sequences derived from gene libraries. Introduction of multiple copies of a single gene sequence into a fertilized egg, raises the probability of integration of the novel sequence into the recipient genome from the infinitely low, if only one copy were introduced, to over 10% of surviving progeny if millions of copies were simultaneously introduced. Thus over the last 7 years the procedure for the production of transgenic animals has become realizable. The technique was strikingly demonstrated by Palmiter et al. (1982) in the

THE APPLICATION OF GENE MANIPULATION TO AQL’ACULTURE

5

mouse, following some earlier initial attempts (Brinstet et al., 1981). Subsequently similar approaches were finding success in the amphibian Xenopus lueok (Etkin and Balcells, 1985), in the fruit fly Drosophila (Spradling and Rubin, 1983), and eventually in other mammals such as rabbits, sheep and pigs (Hammer et al., 1985 ) . In recent years a number of laboratories around the world have become interested in the application of the methodology to various species of finfish. For example , Maclean and Talwar (1984)) and Maclean et al. (1987a,c) have produced transgenic rainbow trout (S&no guirdneri), as also have Chourrout et al. (1986) and Guyomard et al. (1988). Fletcher et al. (1988) and Rokkones et al. (1988) have induced transgenism in Atlantic salmon (Salmo solar), Brem et al. (1988) in tilapia (Oreochromis niloticus), Ozato et al. (1986) in the medaka (0ryzia.s lapites), Dunham et al. (1987) in the channel catfish (Ictulurus sp.), Stuart et al. (1988) in the zebrafish (Brachydanio rerio), and Zhu et al. (1985,1986) and Maclean et al. (1987a) in the goldfish (Carussius aurutus), loach (Misgurnus unguilficaudutus), and other species of freshwater fish. A variety of genes were used in these transfers and varying degrees of success were obtained, of which further discussion will follow. It is appropriate to stress at this point that the initial success of Palmiter et al. ( 1982) in producing mice with enhanced growth rate and increased size was due, not to the fact that a rat growth hormone gene had been introduced into the transgenic mice, but that the novel growth hormone gene from the rat was expressed as growth hormone polypeptide in the liver cells of the recipient mice. Thus a hormone usually produced in small amounts in the pituitary gland was now being made in significantly greater amounts in the liver, as a result of the growth hormone gene coming under the influence of the mouse metallothionein promoter with which it was spliced. This promoter region regulates the expression of the metallothionein coding sequence in liver, by attracting RNA polymerase molecules and transcription factors necessary for transcription of the gene into the relevant messenger RNA; so using the right promoter ensured a successful outcome to these early transgenic experiments. ESSENTIAL STEPS IN TRANSGENIC FISH PRODUCTION

The following are the elements

of producing

transgenic

fish, listed stepwise.

1. Acquiring the gene Acquisition of an appropriate gene sequence from a gene library, together with regulatory flanking sequences is the first step. Flanking sequences should include promoter sequences upstream from the coding sequence and tissuespecific enhancer sequences which may lie up- or down-stream from the coding sequence. It is normally though best to use a genomic rather than a complementary DNA sequence, and thus all the introns will be included in the se-

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N.MACLEANANDD.PENMAN

quence together with substantial flanking sequences up- and downstream from the gene. If a different promoter sequence is to be used - and in work in fish a fish-derived promoter sequence is highly desirable - then this must be inserted upstream of the coding sequences in place of the normal promoter. 2. Cloning of the gene The acquired gene sequence or construct must now be cloned into a plasmid or phage vector and grown up in a suitable bacterial strain. This is followed by harvesting of the gene from the microbial cells and, if desired, isolating it from the vector by the use of appropriate restriction endonuclease enzymes. The purified sequence should be run in an assay to verify its correct molecular weight. 3. Injecting the gene Many millions of copies of the linear sequence should now be available for introduction into the fertilized eggs of the chosen fish species. Some laboratories have chosen to inject circularized sequences complete with the plasmid vector. There are arguments for and against such a procedure. Circularized DNA is almost certainly more resistant to enzymatic degradation in the cell, but is less available for recombinational insertion into the genome of the egg. Viral insertion sequences are sometimes employed at either terminus of the injected sequence with a view to increasing the efficiency of incorporation, but there is little evidence that this further addition to the construct is necessary or beneficial. The insertion of the genes would be ideally carried out by their precise placement in the egg nucleus, but although this is readily possible with mammalian eggs it has proved difficult with fish eggs. Ozato et al. (1986) successfully injected cloned genes into the large oocyte nuclei of the medaka, however. Thus it is usually necessary to place the sequences in the cytoplasm of the egg in the vicinity of the nucleus. In very yolky eggs such as those of fish, it is useless to inject into the yolk itself, and so precise location of the injected DNA solution is vital. Frequently the genes are introduced by micromanipulation using fine sterile microneedles to penetrate the egg chorion. There is evidence from some laboratories that salmon eggs are best injected via the micropyle, but trout eggs are readily injected through the chorion, soon after fertilization. Small eggs with soft chorions, such as those of carp, loach or zebrafish, can be usefully dechorionated before injection. Embryonic development can proceed normally in these species in the absence of the chorion (Stuart et al., 1988). 4. Handling injected eggs Methods for keeping fish eggs after DNA injection do not differ from those used normally. It is of course essential to keep equivalent batches of control eggs to determine the comparative survival rates of the various procedures.

THE APPLICATION OF CEDE MANIPULATION

TO AQUACULTURE

7

Some of these are discussed in our other presentation at this meeting (Penman et al., 1990). Following hatching, the question arises whether transgenic induction should be monitored by sacrificing the fry for DNA samples, or whether they should be grown on to a size at which biopsy may be safely attempted. Most laboratories have chosen to do both, that is, to kill a proportion of fry, and to allow a proportion to grow on. It will be appreciated that if DNA is purified from a complete hatched fry, or even more if recovered from a batch of fry, it is not always possible to distinguish between total transgenism and chaemeric transgenism in the fish. If the novel genes have been incorporated some time after the onset of cell division, it is certain that only those tissues developing from the cell or cells in which incorporation occurred will carry copies of the novel inserts. Such animals which are transgenic in only some tissues are know as chaemeric (or mosaic) transgenics (see, for example, Stuart et al., 1988). You may begin to suspect the presence of chaemerics amongst your transgenie progeny if some give a very weak hybridization signal. A carefully run series of standard blots including some in which the total number of sequences would be equivalent to less than one per cell, enables the possibility of such weak signal blots being a result of chaemerism to be established. In order to determine chaemerism beyond doubt, larger fish can be sacrified and each tissue separately monitored for evidence of transgenic integration. In our own work with transgenic induction in rainbow trout we have discovered some chaemeric fish in our experimental batches. 5. Assaying for transgenism The standard procedure for identifying transgenism is to purify DNA from sacrificed fry or biopsy material and to run dot or slot blots of this DNA, one blot per fish. If the DNA is from biopsy material the fish must of course be tagged, and that is certainly one of the difficulties of transgenic work, since it is not easy to find entirely satisfactory tagging methods for small fish. Blotting simply consists in using a radioactively tagged probe of the novel injected sequence in hybridizing conditions, followed by washing of the blots and autoradiography. But many fish may be positive on dot blots and not be true transgenies, since such an assay only indicates the persistence of one or more copies of the injected sequence, not its genomic integration. It is therefore essential to follow dot blotting by proper Southern blotting of positive samples. Such Southern blotting, to be effective, must depend on possession of detailed information about cleavage sites for specific restriction enzymes along the length of the novel sequence. In other words, a restriction map must be known. Such a map permits accurate determination of predicted sizes of DNA fragments after digestion with a chosen restriction enzyme has gone to completion. This should be checked by loading onto separate lanes DNA samples of the linear injected fragment before and after digestion. The crucial evidence

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for integration, which has not always been provided in work with attempted transgenic induction, is to demonstrate that the terminal fragments of a sequence are of higher molecular weight in the D%A of transgenic fish than they are in the uninjected control DNA. This indicates that these terminal fragments are now spliced to genomic DNA, the section of native DNA thus increasing the molecular weight of each outer restriction fragment of the original integrated sequence (see Fig. 1). (There is an important caveat to the statements in the last two sentences, namely, that injected sequences may on occasion form persisting unintegrated concatamers in head-to-tail, head-to-head or tail-to-tail arrangements, or such recombinant sequences may be integrated. It is not always easy to exclude the possibility of such concatamer persistence, although inheritance via sperm probably does so.) A frequent finding in all transgenic animals, and fish have proved no exception to this, is that some of transgenics have multiple copies of the injected sequence integrated. Some of these copies may be incomplete, others tandemly arranged in the fish genome, so that some quite complex patterns of restriction fragments can result from electrophoresis of restricted DNA from transgenic fish (Penman et al., 1988), But provided one copy can be demonstrated to be entire there is probably no need to be concerned about multiple-copy integration. It is perhaps remarkable that transgenic animals do not display aberrations because of deletion of other DNA when the novel sequence was incorporated. Such incorporation is presumed to occur by by recombinational exchange with some existing piece within a chromosome. Presumably the genomes of eukaryotes are so large that much of the DNA is essentially redundant and therefore random pieces of up to 10 kilobases can be lost without any genetic implication. The genome size of a bony fish such as a trout is about 5 picograms per nucleus, providing enough DNA in a haploid state for up to 1 million genes, yet it is likely that no more than 50 000 separate genes are present in any organisms (extrapolating from data on mutation rates). Of course some deletions by transgenic incorporation may be fatal and contribute to the reduced viability of injected eggs and embryos. But it is not presently evident that transgenic animals commonly carry genetic abnormalities. 6. Is the gene expressed? Once transgenic fish are being produced, the next significant question is whether the novel gene is being transcribed into RNA and that RNA translated into protein. Expression can be monitored by assaying the messenger RNA of transgenic tissues, but normally evidence of expression is sought immunologically at the protein level. Ozato et al. (1986) have demonstrated expression of chicken crystallin genes in their work with the medaka. The choice of promoter is all important where expression is being considered, and Guyomard et al. ( 1988) failed to detect expression in their transgenic rainbow trout using a

THE APPLICATION OF GENE MANIPULATION TO AQUACULTURE

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mouse metailothionein promoter, even after attempted heavy-metal induction (the mammalian metallothionein promoter, as well as conferring high expression in liver, also has sites within the sequence which respond positively to induction by corticosteroid hormone or heavy metals such as zinc and cadmium). A trout metallothionein gene has now been isolated (Bonham et al., 1987; Zafarullah et al., 1989a) and these authors report that the mammalian sequence is expressed 20-fold less than the native fish metallothionein sequence when these genes are transfected into trout hepatoma tissue culture cells (Zafarullah et al., 1989a,b). So it looks as if we will all have to chase genuine fish promoters if expression is to be maximized. The expression of a chicken crystallin gene in the medaka is interesting (Ozato et al., 1986) although a slightly anomalous pattern of tissue expression was obtained with this sequence. Rokkones et al. (1988) report immunological evidence of expression of human growth hormone in transgenic Atlantic salmon. When transgenic induction was first attempted there were fears that the novel sequences would not be expressed because of methylation of their C bases by endogeneous methylase enzymes, or that their integration into abnormal chromosomal locations would lead to aberrant or no expression. Neither of these early fears has proved to be well founded. Position effects seem to be relatively rare and methylation either does not occur or does not silence the expression of the new gene. 7. Are the transgenic fish germ-line transformed? If the transgenic fish have resulted from introduction of a novel gene into the genome of the fertilized egg cell, all tissues should carry at least one copy of the novel sequence in each cell. This implies that the gonads will also be transgenic. If the fish are transgenic heterozygotes with integration at only one locus, then 50% of all sperm or egg cells produced should carry the novel sequence and thus, in a cross with a non-transgenic fish, 50% of all progeny should be heterozygous transgenics. If transgenic fish have more than one entire copy integrated, but at different chromosomal locations on distinct chromosomes, then a complex ratio will be recovered in the progeny, with more than 50% total transgenism. There would be good grounds for wishing to produce homozygous transgenics, and if eggs from transgenic females were treated to gynogenetic induction, then 50% of the progeny should be homozygous transgenic females (by first cell division inhibition from a single copy heteroZYkTOUS ? ) *

Evidence for transmission of the transgenic trait to progeny has already been obtained by Zhu et al. (personal communication, 1989) in the loach ML+ gurnw, Stuart et al. (1988) in the zebrafish and Guyomard et al. (1988) in the rainbow trout. In our laboratory we have assayed gonads of two transgenic fish for the presence of the injected sequence and found these to be positive; there

12

seems to be grounds for optimism sequences to progeny.

N.MACLEANANDD.PENMAN

for the effective

transmission

of inserted

THE CHOICE OF GENES FOR AQUACULTURE TRANSGENICS

It must be stressed that these are early days in the history of transgenism in aquaculture and some of the work undertaken is not primarily designed to have an immediate practical application. Thus much can be learned about fundamental aspects of gene regulation in higher organisms by putting novel genes into fish. But it is equally certain that genes such as metallothionein and growth hormone have been chosen in part because of the possible advantageous contribution to aquaculture of the transgenic animals produced. We consider these potential contributions below. 1. Growth hormone (somatotropin) genes Growth hormone is a polypeptide hormone synthesized in the anterior portion of the pituitary glands of all vertebrates, from which source it is released into circulation and exerts stimulating influences over growth and development. Being a protein rather than a steroid hormone means that it is broken down in the gut if fed to animals, at least in most vertebrates, although in many fetal animals and young stages of some fish species it may pass through the gut and exert influence via the dietary route. Like other polypeptide hormones, such as insulin, it is effective when injected, and it has been demonstrated that avian and mammalian growth hormone is effective in salmonid fish following injection (Gill et al., 1985). In most of the work done to date, mammalian growth hormone genes have been spliced to a mammalian metallothionein promoter before insertion into fish eggs (see for example, Zhu et al., 1986; Dunham et al., 1987; Maclean et al., 1987a; Guyomard et al., 1988). This gene construct is used in the hope that the gene will be expressed continuously at relatively high frequency by the liver cells, rather than intermittently and at low frequency by the pituitary gland cells. We can only speculate on the probable effects on fish growth and development. Fish are known to exhibit great plasticity of growth. For example, fish can be of relatively small size if kept in conditions of restricted diet even if circulating growth hormone levels are high. A super abundance of circulating growth hormone might induce sterility or other side effects, and some of these have been reported as infrequent but detectable events in the work with transgenic mice (Palmiter and Brinster, 1986). Rather than use a growth hormone gene from mammalian source, it might be desirable to inject instead copies of a cloned piscine growth hormone gene. Such sequences have been isolated from Atlantic salmon (Johansen et al., 1989), chum salmon (Sekine et al., 1985), and rainbow trout (Agellon et al., 1988). It is uncertain whether it would actually be more effective than the

THE APPLICATION OF GENE MANIPULATION TO AQUACULTURE

13

mammalian sequence, and if used for injection it would have to be spliced to a separate ‘reporter’ gene to make transgenic detection feasible. Mice transgenic for growth hormone genes expressed in liver become much larger than normal mice (Palmiter et al., 1982 ), and humans who over-produce growth hormone are of giant size. It has been reported that pigs transgenic for growth hormone genes (expressed in liver) showed little acceleration in growth, but it seems probable that the selection for growth rate that has already occurred in intensive pig breeding has used up any simple effects of increased growth hormone production. It seems likely that, with many species of fish, expression of growth hormone genes in liver might yield improved animals for intensive fish farming. 2. Growth hormone releasing-factor genes Growth hormone (somatotropin) releasing factor is a protein which stimulates the cells of the anterior pituitary gland to release increased amounts of somatotropin. Fish transgenic for this gene sequence would be likely to release enhanced amounts of native hormone. A number of laboratories are considering using such a sequence for transgenic induction but results are not yet known. 3. Metallothionein genes Metallothioneins are proteins that bind heavy metals in cells, particularly cadmium, copper, zinc and mercury. They have a dual function in the animal cell. One, and probably the primary function, is to supply zinc to enzymes within the cell that are zinc requiring. Another key function is detoxification, a process which involves binding of these heavy metals within the cell by the protein, followed by excretion of the metallothionein protein complex from the cell and the organism via the kidney. Metallothioneins are also inducible proteins, and their synthesis is greatly accelerated in the presence of heavy metals. Thus fish and other animals, when exposed to a sub-lethal dose of a heavy metal such as cadmium or copper, are able to protect themselves by increased metallothionein synthesis (Maclean et al., 1987b; Woodall et al., 1988). Now metallothioneins have become best known in gene manipulation for their promoter sequences (discussed separately below) but they are interesting genes in their own right since it seems possible that fish or other aquatic animals, with increased numbers of these gene sequences, could better survive temporary heavy-metal pollution. Since heavy-metal toxicity is important in many cases of transient pollution, it will be clear that these sequences may have something to offer. Our own laboratory has already been active in using these sequences for transgenic induction. Sadly metallothionein does not bind or protect against aluminium, so it offers no substantial help for fish in highly acidified waters suffering from aluminium toxicity.

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N.MACLEANASDD.PENMAN

4. Antifreeze genes A number of species of fish living in Arctic or Antarctic seas have evolved proteins in their blood which reduce the effective freezing point of the blood and tissues and so act as natural antifreezes. One fish with such proteins is the winter flounder (Pseudopleuronectes americanus) (Scott et al., 1985; Fletcher et al., 1986). The appropriate genomic sequence has now been isolated and successfully introduced into the Atlantic salmon (Fletcher et al., 1988; Davies et al., 1989). Atlantic salmon possess no natural antifreeze proteins and indeed freeze to death in sea cages when the temperature drops below - 0.7 ‘C (Saunders et al., 1975; Fletcher et al., 1988). This application of gene manipulation technology to a known aquaculture problem is clearly an exciting development and an indicator of what will surely lie ahead in the future on a larger and wider scale. It will probably be some time before the results of this interesting work can be assessed on a commercial scale. There are presumably other aquaculture situations in which antifreeze protein genes could find useful application, but it is no doubt correct to assume that, although these proteins may enable fish to survive in very cold water, they would not be likely to confer cold tolerance on warmwater fish. Interestingly, the winter flounder gene has been successfully introduced into the genome of the fruit fly Drosophila (Rancourt et al., 1987). These authors report that the gene was not expressed when under the control of its own promoter, but was expressed when spliced to a Drosophila heat-shock gene promoter. 5. Crystallin genes These genes code for proteins that are made only in the lens of the eye. Ozato et al. (1986) have introduced a chicken crystallin gene into the small fish, the medaka, and studied its tissue-specific expression in the fish under the control of its own promoter. Their work is of interest in terms of gene regulation, since it demonstrates that a chicken gene promoter is recognized satisfactorily in a fish. 6. Esterase genes Esterases are protein enzymes which hydrolyze esters. In insects it has been found that mosquitoes or aphids which have become resistant to some insecticides have done so by natural amplification of their esterase gene copy number (Field et al., 1988) and have thus been able to break down the insecticide more efficiently. It is conceivable that situations could arise where it would be an advantage to confer on fish an enhanced resistance to insecticide-like compounds, for example in the treatment of sea-caged salmon with therapy against crustacean parasites.

THE APPLICATION OF GENE MANIPULATION TO AQUACULTURE

1.5

7. Disease resistance genes It is likely that in the long term the greatest single contribution that gene manipulation will make to aquaculture will be in terms of increasing the disease resistance of aquatic organisms. Many specific diseases still bedevil the aquaculture industry and disease resistance is in many cases known to depend on possession of specific genes. Unfortunately none of these genes has as yet been identified and the rearing of transgenic fish with enhanced resistance to specific diseases remains only an intriguing possibility for the future. 8. Regulatory gene sequences All genes are, at least in part, under the control of other DNA sequences. All eukaryotic genes have promoter sequences upstream from the transcription start site, and many also have specific enhancer sequences lying further upstream or downstream. Promoter sequences are necessary for proper transcription to take place and often enhancer sequences regulate specific rate control in particular tissues. Some promoter sequences can themselves be activated by specific molecules or factors such as steroid hormones, heavy metals, or heat shock. Since particular genes can be spliced to novel promoters, there is considerable scope for beneficial gene manipulation in the production and injection into organisms of such potentially fruitful combinations of regulatory sequence and coding sequence. (a) The metallothionein gene promoter. This promoter, as has been already remarked, is specifically inducible by both corticosteroid hormone and certain heavy metals such as cadmium or zinc. Any coding sequences combined with it will also be induced by these agents. As a relatively universal promoter, it is active in most tissues, but is particularly active in liver cells. Metallothionein promoters are sometimes species specific, or at least phylogenetic class specific, and thus fish metallothionein promoters will be especially valuable. The splicing of a salmon growth hormone gene to a trout metallothionein promoter and the injection of this construct into salmonid eggs is an obviously interesting scenario for the future. (b) Heat-shock gene promoters. Heat-shock genes are genes specifically inducible by heat shock, and code for proteins which protect cells against the deleterious effects of increased temperature. All organisms have such genes and the sequence which responds to temperature elevation is the upstream promoter. Genes coming under the control of such promoters would be specifically activated by an elevation in temperature. Such promoter sequences could prove useful if it were desirable to have a trans gene which would only be synthesized on specific stimulation of the organism by temperature shock. (c) Other tissue-specific promoters. There is now abundant

evidence that many

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N.MACL.EANk\7lD.PENMAN

regulatory sequences associated with specific protein coding genes confer on these genes tissue specificity of expression. This, taken together with the fact that genes can be separated from their own normal regulatory sequences by manipulation and recombined with others, implies that a wide range of selected tissue specificity of expression will become increasingly available to those interested in transgenic induction. At the same time there is already evidence that regulatory regions active in one species will not always be active in cells of another species, especially if the two species are separated by wide taxonomit distances. It therefore seems that promoter and other regulatory sequences will have to be chosen with care. SOME OTHER POSSIBLE GENETIC MANIPULATION

Beside considering the matter of which genes to employ and which regulatory regions with which to combine these coding sequences, a number of other genetic manipulations are currently possible or probable. One is the use of reporter genes. As previously mentioned, if cloned copies of genes native to a species are injected into an egg and become incorporated in the DNA, some foreign ‘reporter’ sequence must be attached to allow assay for the presence and persistence of the extra copies of the native gene. These could be noncoding sequences of viral DNA; but an additional stratagem is available, namely, the combination of the native sequence with, say, the galactosidase coding region from the bacterium E. coli. This region, if expressed, will yield the enzyme galactosidase, for which there is a highly specific calorimetric test. Galactosidase is a foreign sequence that can be used to reveal the presence of the novel construct, its transcription into RNA, and its translation into protein. A second possible stratagem, and one which has already been successfully used in the mouse (Breitman et al., 1987), is to utilize a gene coding for a protein which, because of its interference with a metabolic pathway, is lethal to the cells in which it is expressed. If such a protein were combined with tissue regulatory regions, specific, for example, for ovary, sterile female fish with no gonadal development might be produced. Thirdly, technology might become available to construct nutritionally crippled strains of fish, requiring, for example, a particular amino acid which the wild species synthesizes constitutively. Provided this amino acid were added to the diet the fish would thrive, but if they escaped into the wild and became dependent on natural food, shortage of that amino acid in the natural diet would result in their failure to thrive, thus eliminating the threat to native gene pools. USE OF FISH AS PRODUCER SYSTEMS

Experiments are already well advanced to use farm animals as a source of valuable pharmaceutical products. Thus Clark et al. (1987) and Simons and

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Clark (1988) have produced transgenic sheep that synthesize human clotting factor IX, with the interesting additional trait that the protein is released into milk since the coding sequence has been spliced to the regulatory region of the ovine lactoglobulin gene (lactoglobulin is a major milk protein). A similar manipulation could conceivably be carried out with fish, e.g., the production of human insulin in rainbow trout liver. The economic comparisons are complex but the idea is obviously interesting. CONTAINMENT AND PLANNED RELEASE

The accidental introduction of foreign crayfish species to European river systems and of escaped salmon from sea cages into lakes and rivers emphasizes the need to minimize ecological disturbance. Transgenic fish or other genetically manipulated aquatic organisms should not be held in conditions where they can escape into natural waterways. Notwithstanding this need for caution, since the rainbow trout has failed to establish itself in any British lake or river, release of transgenic rainbow trout in the United Kingdom, whether by accident or intention, presumably carries no environmental hazard. Although the use of genetic tags of novel but non-coding DNA seems legitimate and acceptable as an experimental tool, even with wild populations, it will be some time before this procedure is accepted as entirely appropriate. Given this necessary caution, it is evident that all the early application of transgenism to widespread aquaculture must involve either sterile fish such as triploids, or species whose escape poses no threat since they have been proved to be incapable of hybridization or colonization. THE FUTURE OF TRANSCENIC INDUCTION FOR AQUACULTURE

It seems almost certain that the introduction of novel genes into aquatic organisms will make a major contribution to aquaculture, just as it is doing in both plant-based and animal-based agriculture. Aquaculture has special problems and sensitivities, such as the ease of escape, and the important public image of aquatic animals as wild creatures. But against this must be set some cautious optimism about improving the organism’s table qualities, health, and profitability for the producer. When we consider the range of genetically affected traits which are included in these last three parameters - growth rate, colour, flavour, texture, resistance to disease and tolerance of intensive culture - it is hard not to see a bright future for transgenic organisms in aquaculture. But we suggest the laboratory activities must move on, from simply using the available genes that have been cloned by others, to a more fundamental approach involving the construction of genomic libraries from a range of finfish and shellfish species and the isolation of useful sequences from these genetic storehouses.

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ACKNOWLEDGEMENTS

We acknowledge financial support from the Natural Environment Research Council for work from our own laboratory mentioned in this review.

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