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Journal of Biotechnology, 16 (1990) 155-170
Elsevier BIOTEC 00553
Minireview
Transgenic farm animals F. G a n n o n
1,2, R.
Powell 1, T. Barry
2, T.G.
McEvoy 3 and J.M. Sreenan 3
1 National Diagnostics Centre/BioResearch Ireland, University College, 2 Department of Microbiology, University College, and 3 Teagasc, Agriculture and Food Development Authority, Belclare, Tuam, Galway, Ireland
(Received 1 December 1989; accepted 5 April 1990)
Transgenics; Farm animals; D N A ; Inheritance
Introduction Since man became a farmer, he has constantly attempted to improve the quality and productivity of his stock. This was required originally for survival. N o w it is commercially imperative. Traditional methods of breeding based on selection of animals with desirable characteristics, have been assisted for some time by the use of artificial insemination and the statistical analysis of relevant parameters to yield improvements in a consistent manner. Although these methods are powerful and successful, they are, by their nature, gradual. The current economic and consumer demands suggest that quantum changes in the productivity and quality of domestic animals are needed. Examples of this are the public awareness of the deleterious effects on health of animal fats and the consequences of quota systems for animal products in the European Community. Methods developed in the early 1980s suggest that dramatic changes in an animal phenotype can now be achieved within a single generation. Briefly, these procedures involve: (a) The isolation of a gene which codes for a product of potential interest either per se or because of its effect on animal performance; (b) The preparation of a D N A construct which will allow the gene to be expressed in the tissue(s) of choice, thereby by-passing the metabolic controls which normally exist in the animal; Correspondence to: F. Gannon, National Diagnostics Centre/BioResearch Ireland, University College,
Galway, Ireland. 0168-1656/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
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(c) The transfer of the DNA construct in vitro to a recently fertilized 1-cell ovum; (d) The reimplantation of the treated ovum into suitable recipient and (e) The analysis of the offspring. If the DNA construct has been integrated into the chromosome of the animal it is called a transgenic animal. Clearly the desired outcome from this protocol is the beneficial expression of the novel gene in the transgenic animal. As the injected DNA is integrated into the genome of the animal it should be transmitted in the germ line to the next generation. In this way very significant changes in the characteristics of animals may be achieved. Experiments of the type outlined above were first successful in mice (see below) and in recent years have been extended to other animals. This work is motivated by the need to: (a) Increase the efficiency of food conversion in domestic animals; (b) Generate animals with improved characteristics, and (c) Use animals as production units for compounds of therapeutic interest. In this review all three aspects will be considered as will the information obtained from laboratory animals which is pertinent to the development of transgenic commercial animals. The major question facing those who have recognised the potential of transgenics is whether or not they should be generated. This question will be addressed in the closing section of the review.
Transgenic mice Widespread public and scientific interest in transgenics was first aroused when the cover of Nature carried a photograph of two mice one of which was twice the size of its litter mate. The increased growth was a consequence of the insertion of a growth hormone gene into the pronucleus of a fertilized ovum (Palmiter et al., 1982). The generation of the 'super mouse' was the culmination of many years of effort in the relevant technologies. As is clear from the outline of the transgenic animal production method, a good understanding of how to manipulate embryos in vitro, how to microinject DNA with precision, how to make DNA constructs which can be expressed in animals, and how to reimplant ova into appropriately synchronised recipients were prerequisites for this dramatic demonstration of the technique. This particular experiment, while not the first successful attempt to make a transgenic animal (see for example Gordon et al., 1980; Constantini and Lacy, 1981; Gordon and Ruddle, 1981; Palmiter et al., 1982b) nonetheless indicated the awesome power of the technique. Some details of relevance to the generation of transgenic commercial animals will now be considered. Reviews which provide more detailed information on the topic of transgenic mice include those by Palmiter and Brinster (1985, 1986).
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Method of introduction of D N A into ova
A fertilised ovum is an intact and relatively impermeable entity. Methods had to be developed which would permit transfer of foreign D N A across this barrier. These methods are of two kinds; physical and biological. Currently the most successful approach is the physical microinjection of the D N A into the pronucleus of the ovum. Although requiring undeniable skill and specialist equipment, the large number of transgenic mice that have resulted from this method indicates that it is a reliable technique. For some time the only biological method available was the use of retroviruses as vectors of DNA. Embryos exposed to these vectors can become infected, allowing the DNA of interest to be integrated into the genome. However, this approach has several disadvantages: - The use of retroviruses is unlikely to be acceptable for the production of transgenic animals for human consumption; - Special containment facilities are required to ensure that safety norms are maintained; - Complicated host-vector combinations are a prerequisite to the use of retroviruses which are, of necessity, incapable of self replication; - When integration occurs, the single copy of the gene of interest that is inserted, is usually flanked by the long terminal repeats of the retrovirus which can interfere with expression; - Integration occurs when the embryo is already at a multi-cell stage of development with a consequent diminution of its effect on the founder animal and on the initial efficiency of germ line transfer. For these reasons retroviruses are not widely considered as a practical option in the long term for the delivery of DNA although they have been used successfully (for example Jaenisch, 1976; Soriano et al., 1986; Bosselman et al., 1989). Very recently a new biological alternative has been reported which combines efficiency with simplicity. In essence, the sperm of the animals is bathed in the D N A of interest and, by mechanisms that are not fully described, carries it into the ovum during in vitro fertilisation (Lavitrano et al., 1989). The efficiencies obtained for the generation of transgenic mice was 30% compared to 25% by the more complicated procedure of microinjection (Brinster et al., 1985). This new method is now under active consideration in other laboratories and if successful it is clear that it will be widely used for many different species.
Integration of foreign D N A into the g e n o m e
When the microinjection approach is used, several hundred copies of the D N A of interest are usually introduced into the pronucleus. Successful generation of transgenics demands that at least one of these be integrated into the genome. The mechanism by which this occurs is obscure at the molecular level. The data available show that the number of DNA copies which are intergrated and the sites of
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integration are variable. Usually several D N A copies are integrated in tandem in a head to tail fashion but other arrangements also occur (Palmiter et al., 1983). A hypothesis has been proposed whereby DNA integration occurs at random breaks in the chromosomal DNA (Brinster et al., 1985). These breaks are the rate-limiting step for integration. Interaction between the ends of the injected D N A and the chromosomal break points allows its insertion into the genome. This may explain the frequently observed lower efficiency of integration when using circular rather than linear DNA molecules (Brinster et al., 1985). The tandem array may then be formed either by homologous recombination between the inserted D N A and other injected DNA molecules, by ligation of the injected material in the pronucleus prior to integration or by amplification of the D N A when inserted into certain chromosomal locations. No evidence for any site-specific integration or of recombination between injected DNA and homologous mouse sequences has been found. Analysis of many integration sites may in time reveal more information on these points. However already, deletions, duplications and short novel sequences, found neither in the injected DNA nor in the area of integration, have been found at integration sites (Woychik et al., 1985).
Expression of transgenes Although the integration of foreign D N A in the genome can be demonstrated by techniques such as Southern blotting, a positive result from such analysis is academic if the inserted gene is not expressed. Our knowledge of the requirements for transcription and translation in eukaryotes is advancing rapidly and now includes roles for a variety of transcription factors which interact with the region 5' to the gene for maximum and controlled expression (see Dynan, 1989 for example). However, when transgenic mice were first produced it was unclear whether the mere provision of promoter and related sequences would be sufficient to allow expression of the gene. It was possible that: - The transgene would integrate in the 'incorrect' locus in the chromosome and this would override other influences in transcription; - The promoter region defined in vitro or in transient assays in cell culture might be inadequate in vivo; - The factors required for expression might be limiting; or - The hybrid gene might lack some subtle combination of sequences required for the efficient transcription of natural genes. Because of transgenic animal experiments, we now know the answers to many of these questions. The successful expression of sequences inserted into transgenic animals has shown that the incorrect chromosomal location does not necessarily prevent expression. However, it is frequently alluded to in explanations of the varying levels of expression obtained with different transgenics that contain the same DNA construct. The promoter regions used are frequently very short and they obviously are effective. Occasionally in vitro studies are not completely accurate in
159 their description of the requirements for transcription. One such example is the fact that sequences approximately 10 000 bases from the rat albumin promoter enhance expression in transgenics whereas such influence was not recorded during in vitro or in transfection studies (Pinkert et al., 1987). It is impossible to directly test for the adequate availability of transcription factors but one indication of a possible limitation is the fact that there are not normally correct gene dose effects in transgenic animals i.e. although a variable number of copies of the D N A are inserted into the genome, the product level in the animal does not relate to that number (see for example Palmiter et al., 1982a). This could be due to chromosomal location; however, when transgenic progeny are analysed, it appears that an alteration in the gene copy number does not always change the level of product. Because strong promoters are the usual choice for DNA constructs used in transgenics, it is possible that some factors required for efficient transcription are depleted when this extra demand is placed on the cell.
Tissue
specificity
of
expression
The mouse metallothionein promoter was used in the initial constructs which resulted in extremely high levels of growth hormone and large mice. It had been chosen because it is expressed in the liver and could be modulated by heavy metals and glucocorticoids. These controls were less than absolute in the transgenics and were of little practical importance. The control by zinc, however, did indicate that transgenic animals could maintain control features present in the promoter when it is not in its natural locus. An obvious extension of this was the demonstration that tissue specificity can also be achieved with constructs used in the generation of transgenics. Some promoters have a very restricted range of cell types in which they will function. Examples of these include elastase (Ornitz et al., 1985) and insulin (Bucchini et al., 1986) that are expressed in the pancreas or the crystallin gene (Overbeek et al., 1985) which is expressed in the eye. Transgenic mouse experiments (Swift et al., 1984; Seldon et al., 1986) showed that these and many other promoters act in an appropriate tissue-specific manner (Palmiter and Brinster, 1985). In some experiments, promoters from mammals other than mouse were used and tissue specificity was retained in the transgenic mice. A rapidly expanding literature now shows that the ability to express sequences in targeted cell types is used to: - Define promoter and cell specificity requirements by deletion and mutation experiments; - Examine the consequences of ablating certain cell types in animals (for example see Borrelli et al., 1989); - Describe the susceptibility of animals to expression of different oncogenes in different cells (for example see Brinster et al., 1984); - Assess the impact of increasing the circulating levels of certain hormones on the overall physiology of the animal.
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Inheritance From the design of the transgenic experiment which envisages the incorporation of DNA into the chromosome, it is clear that some of the offspring of transgenic founder animals should also be transgenic. It has been shown repeatedly that 50% of the offspring are transgenic, if the injected DNA integrates into a single chromosomal locus in the founder. Occasionally this does not occur. These cases are most readily explained by a degree of mosaicism in the founder animal (for example see Palmiter et al., 1984) which can occur if the injected DNA is not integrated into the chromosome prior to cell division. In microinjection experiments, with mice, the extent of mosaicism suggests that the DNA can persist in ova for up to three cell divisions. In addition some DNA combinations contain elements which allow for episomal persistence of the injected DNA, but this is not a frequent occurrence (Rassoulzadegan et al., 1986; Leopold et al., 1987). When the DNA fragments are initially inserted in the chromosome, multiple copies of the gene are frequently co-integrated (see above). One of the surprises from the early transgenic experiments was the alteration and editing of these sequences in the offspring. Different explanations have been proposed for this including a selection against the unusual structures which might result from multicopies or some influence on the locus due to inappropriate methylation. Once the editing has occurred in first progeny, the transgenic sequences then appear to be stable and are inherited like other autosomal genes. A final aspect of the inheritance of transgenes is the fact that transgenic animals are frequently less fertile than their non-transgenic litter mates. In this case the gene product and the cell type in which expression occurs are of importance. It has been suggested that a promoter which is active in germ cells diminishes the fertility of the animal. Also the presence of high circulating levels of growth hormone for example, perturbs the physiology of the animal thereby reducing its fertility. However, in most cases, transgenic mice with useful traits can be successfully used to establish colonies which perpetuate these characteristics.
Transgenic farm animals The results of transgenic mouse studies reinforce the idea that this model can be applied to animals of economic importance. The method and skills used to insert DNA into cell ova have been learned by many using the mouse system. The general requirements for D N A constructs which allow expression of genes of interest have been established and the ability to transmit these traits in the germline has been demonstrated. Nonetheless, the generation of transgenic farm animals presents technical difficulties which have not been encountered in laboratory species.
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Methodology of gene transfer in farm animals The choice of method for transfer of D N A to fertilized ova is based on the same reasoning as for mice and currently microinjection is the most widely used approach. This procedure in mice is aided by the fact that the ova are partly transparent and the pronuclei readily visualised. In species such as cattle and pigs, microinjection procedures are more complex because of the high lipid content and the opacity of the ova. This difficulty is now overcome by the use of centrifugation and the DIC (Nomarski) microscopy. Viable ova with discernible pronuclei can be obtained in this way. A second and major limitation when farm animals are used is the difficulty of obtaining large numbers of ova. The yield is increased by gonadotrophin administration but the average number of suitable ova obtained in cattle is typically about seven. The recovery of recently fertilised ova from cattle, sheep and other animals requires full scale surgery following artificial insemination. Then, following D N A microinjection, the ova are surgically transferred to the oviducts of synchronous recipient animals. The logistics and costs of the total procedure are major impediments to the production of transgenic farm animals. This can be further aggravated by the fact that the current expected frequency of production of transgenic calves is very low ranging from 0-2% of calves born from gene injected ova. Alternative methods for generating large numbers of ova required for efficient transgenic farm animal experiments include attempts to establish embryo carcinoma cell lines equivalent to those available for mice (Kuehn et al., 1987). These would be an unlimited source of starting material which could be regenerated into whole animals. An added attraction is the fact that selection of transgenic cells might be possible, by use of appropriate markers, prior to transfer to recipient animals. Another, more advanced development would be the use of in vitro fertilization and in vitro maturation procedures. These involve the use of preovulatory oocytes which can be matured and fertilized in vitro (Lu et al., 1987). Subsequent steps would involve microinjection of the foreign DNA, in vitro selection of ova that contain this D N A integrated into the genome and non-surgical transfer of these ova to recipient animals. Methods for identification of developing ova that have integrated the microinjected DNA will have to be very sensitive as development in vitro of some animal ova is blocked after a small number of cell division. The use of techniques such as the polymerase chain reaction (PCR) which amplifies defined D N A fragments approximately 1 million-fold may facilitate the detection of such ova (Saiki et al., 1988). This procedure therefore is likely to involve the removal of one or two biopsy cells from the developing embryo. These can then be analysed to confirm the presence and integration of injected DNA. The biopsied embryo should develop normally following such treatment as is evidenced by the ability of bisected embryo halves to resume normal development.
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Farm animals with improved characteristics As growth hormone (somatotropin) given in repeated injections to many animals can increase their efficiency of food conversion, growth characteristics and milk yield, it is not surprising that many of the transgenic experiments with farm animals have focussed initially on components in the growth endocrine cascade. The aim of these experiments is not to produce mammoth animals but to provide animals that are ready for slaughter at younger ages and have more effective food conversion. The production of animals with lower fat content, with larger litter sizes, with greater milk yields or with improved disease resistance are further targets for this research. In all of these instances the transgenic method is attempting to rapidly provide animals of a type which might be obtained in due course by the current standard agricultural methods. Work on transgenics can also be directed to altering animals in a way which is not possible by standard methods. It should be possible to change the content of milk by directing the expression of proteins of interest to the mammary gland. The resulting benefits could be nutritional or commercial depending on the protein involved. An example of the latter might be the increased expression of some caseins which might have useful applications when purified. An extension of this thinking would be to alter other biochemical characteristics in the animal. The current medical view is that animal fats are unhealthy. This is due to the presence in them of unsaturated fatty acids. A portion of these fatty acids could be rendered polyunsaturated by the inclusion in animals of the gene which codes for the enzyme delta-12-desaturase which is found in plants. It is estimated that approximately 30% of the animal fatty acids could be made polyunsaturated in this way (Gannon, 1986). An alternative use of transgenic farm animals is as bioreactors which produce therapeutic proteins of high value. Many of these proteins come from man or other mammals originally and are frequently modified by post-translational glycosylation or acetylation. When these genes are molecularly cloned, the choice of host in which they are expressed is frequently bacteria (which do not have the ability to glycosylate), yeasts (which modify proteins in a manner which does not exactly correspond to mammalian cells) or animal cells in bioreactors (Gannon, 1985) which are not yet cost-effective. Transgenic farm animals could be hosts of choice if the levels of expression obtained are economical. In addition, the transgenic animal may be uniquely suited for production of large or multi subunit proteins which need cofactors for their activity.
Expression in transgenic farm animals It is evident that transgenic farm animals could be of value in many different aspects of Biotechnology. One measure of this is the fact that currently over 40 applications are under consideration for transgenic animal patents (Ford, 1988; Fox, 1989). As a consequence, there are relatively few scientific publications which describe work in this area (Pursel et al., 1989).
163 The research reported relates to both animal improvement and protein production by transgenics in a number of species. In all cases a gene of interest must be expressed in a chosen tissue. The required end point has a major influence on the choice of promoter used in the DNA constructs. Typically, researchers involved with modification of the whole animal have selected promoters which function well in the liver as it is the largest internal organ and its products are readily transferred to the blood stream. The mouse metallothionein promoter fulfulls that requirement but also expresses in a wide range of other cell types. This can be an advantage if the product is thought to act as an autocrine but may be a disadvantage if its over-expression disrupts the normal function of sensitive cells. Alternatives to the use of the metallothionein promoter are liver-specific promoters. In this context we have recently isolated the bovine serum albumin promoter for use in transgenic studies (S. Power, T. Barry, and F. Gannon, unpublished). An efficient liver promoter could also be of use where therapeutic products are required because protein purification from blood can be readily performed. However, many of these products could have detrimental effects on the animal (for example over-production and circulation of blood clotting factors or lymphokines). For this reason, examples quoted below will show that promoters which are specific to the mammary gland, from which the product is secreted in milk, have been the choice of most groups who wish to use the animals as bioreactors.
Examples of transgenic farm animals Following the successful generation of the super mouse it was clear that the mouse metallothionein promoter-human or rat growth hormone D N A construct would be used in other species. The first published results following that came from Hammer et al. (1985) using rabbits, sheep and pigs. The results were disappointing (Lovell-Badge, 1985) and showed clearly that the application of transgenic technology to farm animals would not be simple. Low levels of expressed growth hormone were detected in rabbits and pigs but had no effect on growth rate. Numerous attempts have since been made to generate transgenic farm animals and these have been most successful with sheep and pigs. Interestingly the focus of work with sheep has been to use them for expression of high value proteins whereas pigs have been targeted for modification of the whole animal.
Sheep The work with transgenic sheep recently culminated in the expression in the mammary gland of the human blood clotting protein, Factor IX (Clark et al., 1989). The sheep ~-lactoglobulin promoter which was previously shown to be specific for the mammary gland and to express factor IX in mice (Simons et al., 1987) was used. The overall efficiency of the experiment in sheep was approximately 2% illustrating the need for large numbers of animals when embarking on such studies (Clark et al., 1987; Simons et al., 1988). The levels of expression of Factor IX or human
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al-antitrypsin in these experiments were low and cannot yet compete with other expression systems. Work is continuing to optimise the levels of production. There have also been reports on the development of transgenic sheep to over-express epidermal growth factor and other proteins of value. Many of these have not been published in a complete form and are therefore difficult to comment upon. Nonetheless, the impression is created that a number of research groups, usually with commercial collaboration, have succeeded in establishing the technology to generate transgenic sheep.
Pigs In a manner analogous to sheep, a number of groups have reported success in generating transgenic pigs. These have focussed primarily on the over-production of human growth hormone. Brem et al. (1985) were among the first to report the birth of a transgenic pig, after the transfer of 268 zygotes, again underlining the practical difficulties of performing these experiments. Since then, constructs using either the Moloney MuLV promoter (Ebert et al., 1988) or the mouse metallothionein promoter (Vize et al., 1988) have resulted in the over-expression of growth hormone in a small number of pigs, none of which were healthy. A very recent detailed description (Pursel et al., 1989) of a series of experiments with transgenic pigs in which bovine growth hormone levels are elevated showed that individual pigs had increased weight gain but the authors suggest that this should not be over-interpreted because individual controls could also differ from the mean by as much as 30%. When the offspring from two transgenic lines of pigs were analyzed, it was shown that in one case transgenic pigs grew 15% faster than littermate controls if the diet was supplemented with extra protein and lysine. In addition, significant improvements in feed efficiency and a decrease in carcass fat were noted. However, a number of pathologies in the pigs were also described by the authors who believe that these could be diminished by a combination of careful selection of the founder animal and by limiting transgene expression to the rapid growth phase of the animals (Pursel et al., 1989), A number of research projects are suggested by this study including the description of systems that can be controlled by exogenous factors, a complete characterisation of the factors which interact to result in promoting growth, the influence of nutrition on defining an optimal circulating concentration of growth hormones and the consequences of the use of promoters other than metallothionein.
Cattle Many groups are working towards production of transgenic cattle with improved characteristics. Again the published literature provides little in the way of detail. Transgenic calves with improved growth have not been formally reported but reference has been made, without documented data, to calves which express antigenic epitopes of virus (Church, 1987) and to the expression of fl-interferon (see Van Brunt, 1988). The effects on the animals are unknown. This paucity of information contrasts with the major commercial interest in this species and is
165 reflected in background information articles in biotechnology journals (e.g. Klausner, 1988; Van Brunt, 1988) in which ongoing experiments using actin or B-casein as promoters for the expression of the estrogen receptor or growth factors were referred to. Presumably, when patent aspects are clarified, this information will become available. Rabbits
Transgenic rabbits have also been produced. Because rabbit ova are transparent, microinjection is less complicated than for other species. Some basic scientific experiments have been extended from transgenic mice to transgenic rabbits (Knight et al., 1988). Growth hormone has also been expressed in transgenic rabbits (Hammer et al., 1985) and proteins of commercial importance have been produced in rabbit milk (L.M. Houdebine, personal communications). It would appear that the latter is the application which might be of relevance in the future for transgenic animals of this species. Fish and chickens
Although microinjection into the pronucleus is the method of choice for most animals, it is not appropriate for either fish or chickens. For chickens it would appear that retroviruses might provide the best approach given the physical difficulties of working with avian eggs. Salter et al. (1987) and Bosselman et al. (1989) have succeeded in transferring proviral fragments to chickens and demonstrated mendelian inheritance of this DNA. Similar results using drug resistance gene markers have been reported by Nigon (1988). These results clearly point the direction in which research on transgenic chickens will proceed. The challenge to apply transgenic methods to fish which are grown commercially is at first sight simplified by an unlimited supply of eggs, easy in vitro fertilisation and development ex utero. A variation of the microinjection scheme is used with the DNA delivered to the cytoplasm rather than the nucleus (Chourrout et al., 1986). For some noncommercial species microinjection into the nucleus is possible and has been successful (see review McLean et al., 1987). A difficulty in reviewing the literature on transgenic fish relates to the persistence and indeed amplification of episomal injected DNA. To date there are no detailed descriptions of transgenic fish that express the transferred genes (see Powell et al., in press). Inheritance of the foreign DNA has been demonstrated (Guyomard et al., 1989) and occasional reports of expression of foreign genes several weeks after microinjection (Ozato et al., 1986; McEvoy et al., 1988) have appeared but the lack of familiarity with the molecular biology of fish is proving to be a major limitation in the preparation of DNA constructs which will be effective. The recent cloning of the salmon growth hormone gene (Johansen et al., 1989; Lorens et al., 1989) and of a variety of other fish genes (Wolff and Gannon, 1989; R. Powell, L. Byrnes and F. Gannon, unpublished) should provide a more solid basis for these studies in the near future.
166 The future
In a very short period of time transgenics has moved from a futuristic concept to a challenging reality. Man can now alter the genetic characteristics of m a n y animals. At a technical level, work is forging ahead to improve and extend these techniques. Methods to increase the supply of ova, to facilitate the transfer of the D N A constructs to the ova, to establish selection procedures for transgenic ova prior to reimplantation, and to increase the control, specificity and level of expression of selected genes are all under active study. With time it is clear that species for which current methods are inefficient will be added to the repertoire available to scientists. Already much has been learned from transgenic studies which has advanced our understanding of the normal and the pathological in mammals and this will continue. New whole animal test systems have become available to allow harmful effects of compounds to be identified (Fox, 1988). Early examples show that farm animals can be used in the manufacture of complex products of therapeutic benefit. As different construct and animal combinations are prepared it will be possible to make rational commercial decisions on the future of this technology. Finally, animals, fish and birds have been generated which can be considered to be the first in a series which should eventually yield species with improved or altered characteristics. The commercial benefits from this are potentially large. The advent of a potent new technique with clear commercial potential would normally give rise to extravagant extrapolations of the value of the development. This is not happening for transgenics and indeed one analysis of the current climate is that there will be very restricted use of transgenics. There are m a n y converging reasons for this ranging from concern for animal welfare to commercial doubts. When a fragment of D N A is inserted into the genome of an animal the precise outcome is unknown. Harmful mutations can occur, although these are rare. Greater problems are encountered in the context of the altered physiology of the animal due to the over-expression of a potent biochemical. It is now documented that some animals are prone to a variety of ailments when they are transgenics. This is best documented for pigs with increased growth hormone levels (Pursel et al., 1989). It seems reasonable that combinations of D N A constructs and animal will be obtained for many species in which the benefits are not diminished by the side effects. In the meantime want on and unnecessary hardships to animals must be avoided. Any transgenic offspring that have disabilities must be quickly and humanely killed. Concern for animal welfare and mere pragmatism, which recognises that such animals could not be the basis for a commercial venture, demand this. If transgenic farm animals with useful characteristics are made, then a number of other questions must be faced. The practice of using constructs which were shown to be useful in mice for experiments with larger animals still results in the incorporation of D N A fragments from heterologous species or from viruses. It is not helpful for the image of transgenics and, for some, of the ethics of the experiment if the key component originated from man, mouse, rat or viral DNA. For this reason we, and others, have sought homologous promoters and active genes for the purpose of making transgenic farm animals. This trend is certain to continue in the future.
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A further question relates to the free release of recombinant organisms. For m a n y domestic animals the 'release' can be easily controlled. When any p r o g r a m m e moves to the next phase in which the animals are used for breeding purposes, the long-term consequences will have to be adjudicated upon. Those who place the environment at the top of a list of priorities will demand, as a minimum, that great caution be exercised. Given the scientific impossibility to exclude absolutely any negative consequences of free release, it would seem likely that a number of generations of transgenics resulting from diverse cross-breeding experiments will be required for study in well-controlled environments. The resolution of many of the practical problems related to transgenics can be achieved as outlined above. In biotechnology, the step from an ability to make a product to the use of this ability is decided by consideration of commercial criteria. The uncertainty related to transgenics, because of the environental and animal welfare aspects, has a very negative influence on businesses which might become involved in transgenics. The benefits of animals with improved growth rates, food conversion or decreased fat content can be appreciated by those industries, but they have not been presented with a clear example of a healthy transgenic animal with these traits. When this happens the analysis by m a n y companies of the future situation will undoubtedly become more positive. Without industrial investment there is the possibility that the research in transgenics will lose m o m e n t u m and become a laboratory-based technique. National and international funding agencies are currently responding to the anti-transgenic lobby in a way which makes it difficult to promote more research in this area. If transgenics becomes, in time, an example of regulatory consideration impeding a controlled and informed development of a technique, a number of direct benefits will be lost. The therapeutic products from biotechnology which carry the hopes for treatment of many human diseases may in time be most appropriately obtained by expression in transgenic animals. The testing of new products for toxins may in fact be best performed on whole animals that are sensitized to identify the carcinogens rather than in bacteria or in cell culture where only a limited number of cell types and sets of conditions can be tested. The accepted justification for decades of breeding programmes has been to make animals that either produced more milk or meat in a cost-effective manner. This aim of work on transgenics is equally laudable and the prospects for significant progress towards these goals in an accelerated time frame are real. It would seem an error if the balance of scientific potential and of regulatory prudence did not find an agreed basis for further progress in the important area of transgenics. If such a basis is not found then one must wonder which will be the next aspect of science to be obstructed.
Acknowledgements Work referred to in this review includes aspects funded in the laboratories of F. G a n n o n and J. Sreenan by the E.C. Biotechnology Action Programme.
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169 Johansen, B., Christian Johnsen, O. and Valla, S. (1989) The complete nucleotide sequence of the growth-hormone gene from Atlantic salmon (Salmo salar). Gene 77, 317-324. Klausner, A. (1988) What's hot in start-ups. Biotechnology 6, 666-670. Knight, K.L., Spieker-Polet, K., Kazdin, D.A. and Oi, V.T. (1988) Transgenic rabbits with lymphocytic leukemia induced by the c-myc oncogene fused with the immunoglobin heavy chain enhancer. Proc. Natl. Acad. Sci. U.S.A. 85, 3130-3134. Kuehn, M.R., Bradley, A., Robertson, E.J. and Evans, M.J. (1987) A potential animal model for Lesch-Nyhan syndrome through introduction of HPRT mutations into mice. Nature 326, 295-298. Lavitrano, M., Camaioni, A., Fazio, V.M., Dolci, S., Farace, M.G. and Spadafora, C. (1989) Sperm cells as vectors for introducing foreign DNA into eggs: genetic transformation of mice. Cell 57, 717-723. Leopold, P., Vailly, J., Cuzin, F. and Rassoulzadegan, M. (1987) Germ line maintenance of plasmids in transgenic mice. Cell 51, 885-886. Lorens, J., Nerland, A.H., Male, R., Lossius, I., Telle, W. and Totland, G. (1989) The nucleotide sequence of Altantic salmon growth hormone cDNA. Nucl. Acids Res. 17, 2352. Lovell-Badge, R.H. (1985) Transgenic animals: new advances in the field. Nature 315, 629. Lu, K.H., Gordon, I., Gallagher, M. and McGovern, M. (1987) Pregnancy esablished in cattle by transfer of embryos derived from in vitro fertilization of oocytes matured in vitro. Vet. Rec. 121,259-260. McEvoy, T., Stack, M., Keane, B., Barry, T., Sreenan, J. and Gannon, F. (1988) The expression of a foreign gene in salmon embryos. Aquaculture 68, 27-37. McLean, N., Penman, D. and Zhu, Z. (1987) Introduction of novel genes into fish. Biotechnology 5, 257-261. Nigon, V.M. (1988) Progress Report, Biotechnology Action Programme of the European Community 3, 753-756. Ornitz, D.M., Palmiter, R.D., Hammer, R.E., Brinster, R.L., Swift, G.H. and MacDonald, R.J. (1985) Specific expression of an elastase-human growth hormone fusion gene in pancreatic acinar cells of transgenic mice. Nature 313, 600-603. Overbeek, P.A., Chepelinsky, A.B., Khillan, J.S., Piatigorsky, J. and Westphal, H. (1985) Lens-specific expression and developmental regulation of the bacterial chloramphenicol acetyl transferase gene driven by the murine aA-crystalhn promoter in transgenic mice. Proc. Natl. Acad. Sci. U.S.A. 82, 7815-7819. Ozato, K., Kondou, J., Inohara, H., Iwanatsu, T., Wakamatsu, Y. and Okada, T.S. (1986) Production of transgenic fish: introduction and expression of chicken delta-crystallin gene in medaka embryos. Cell Differ. 19, 237-244. Palmiter, R.D. and Brinster, R.L. (1985) Transgenic mice. Cell 41, 343-345. Palmiter, R.D. and Brinster, R.L. (1986) Germ line transformation of mice. Annu. Rev. Genet. 20, 465-499. Palmiter, R.D., Brinster, R.L., Hammer, R.E., Trumbauer, M.E. and Rosenfeld, M.G. (1982a) Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature 300, 611-615. Palmiter, R.D., Chen, H.Y. and Brinster, R.L. (1982b) Differential regulation of metallothionein-thymidine kinase fusion genes in transgenic mice and their offspring. Cell 29, 701-710. Palmiter, R.D., Norstedt, G., Gelinas, R.E., Hammer, R.E. and Brinster, R.L. (1983) Metallothioneinhuman GH fusion genes stimulate growth of mice. Science 222, 809-814. Palmiter, R.D., Wilkie, T.M., Chen, H.Y. and Brinster, R.L. (1984) Transmission distortion and mosaicism in an unusual transgenic mouse pedigree. Cell 36, 869-877. Pinkert, C.A., Ornitz, D.M., Brinster, R.L. and Palmiter, R.D. (1987) An albumin enhancer located 10kb upstream functions along with its promoter to direct efficient, liver-specific expression in transgenic mice. Genes and Development 1,268-276. Powell, R., Byrnes, L. and Gannon, F. (1990) Transgenic Fish. Gen. Eng. Biotechnol., 10, 7-9. Pursel, V.G., Pinkert, C.A., Millar, K.F., Bolt, D.J., Campbell, R.G., Palmiter, R.D., Brinster, R.L. and Hammer, R.E. (1989) Genetic Engineering of livestock. Science 244, 1281-1288. Rassoulzadegan, M., Leopold, P., Vailly, J. and Cuzin, F. (1986) Germ line transmission of autonomous genetic elements in transgenic mouse strains. Cell 46, 513-519. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich,
170 H.A. (1988) Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487-491. Salter, D.W., Smith, E.J., Hughes, S.H., Wright, S.E. and Crittenden, L.B. (1987) Transgenic chickens: insertion of retroviral genes into the chicken germ line. Virology 157, 236-240. Seldon, R.F., Skoskiewicz, M.J., Howie, K.B., Russell, P.S. and Goodan, H.M. (1986) Regulation of human insulin gene in transgenic mice. Nature 321, 525-528. Simons, J.P., McClenaghan, M. and Clark, A.J. (1987) Alteration of the quality of milk by expression of sheep/3-1actoglobulin in transgenic mice. Nature 328, 530-532. Simons, J.P., Wilmat, I., Clark, A.J., Archibald, A.L., Bishop, J.O. and Lathe, R. (1988) Gene transfer into sheep. Biotechnology 6, 179-183. Soriano, P., Cone, R.D., Mulligan, R.C., and Jaenisch, R. (1986) Tissue-specific and ectopic expression of genes introduced into transgenic mice by retroviruses. Science 234, 1409-1413. Swift, G.H., Hammer, R.E., MacDonald, R.J. and Brinster, R.L. (1984) Tissue-specific expression of the rat pancreatic elastase 1 gene in transgenic mice. Cell 38, 639-410. Van Brunt, J. (1988) Molecular farming: transgenic animals as bioreactors. Biotechnology 6, 1149-1154. Vize, P.D., Michalska, A.E., Ashman, R., Lloyd, B., Stone, B.A., Quinn, P., Wells, J.R.E. and Seamark, R.F. (1988) Introduction of a porcine growth hormone fusion gene into transgenic pigs promotes growth. J. Cell Sci. 90, 295-300. Wolff, J.P. and Gannon, F. (1989) cDNA and deduced amino acid sequence of the Salmo salar (Atlantic salmon) adult hemoglobin alpha chain. Nucl. Acids Res. 17, 4369. Woychik, R.P., Stewart, T.A., Davis, L.G., D'Eustadio, P. and Leder, P. (1985) An inherited limb deformity created by insertional mutagenesis in a transgenic mouse. Nature 318, 36-40.
Journal of Biotechnology, 16 (1990) 155-170
Elsevier BIOTEC 00553
Minireview
Transgenic farm animals F. G a n n o n
1,2, R.
Powell 1, T. Barry
2, T.G.
McEvoy 3 and J.M. Sreenan 3
1 National Diagnostics Centre/BioResearch Ireland, University College, 2 Department of Microbiology, University College, and 3 Teagasc, Agriculture and Food Development Authority, Belclare, Tuam, Galway, Ireland
(Received 1 December 1989; accepted 5 April 1990)
Transgenics; Farm animals; D N A ; Inheritance
Introduction Since man became a farmer, he has constantly attempted to improve the quality and productivity of his stock. This was required originally for survival. N o w it is commercially imperative. Traditional methods of breeding based on selection of animals with desirable characteristics, have been assisted for some time by the use of artificial insemination and the statistical analysis of relevant parameters to yield improvements in a consistent manner. Although these methods are powerful and successful, they are, by their nature, gradual. The current economic and consumer demands suggest that quantum changes in the productivity and quality of domestic animals are needed. Examples of this are the public awareness of the deleterious effects on health of animal fats and the consequences of quota systems for animal products in the European Community. Methods developed in the early 1980s suggest that dramatic changes in an animal phenotype can now be achieved within a single generation. Briefly, these procedures involve: (a) The isolation of a gene which codes for a product of potential interest either per se or because of its effect on animal performance; (b) The preparation of a D N A construct which will allow the gene to be expressed in the tissue(s) of choice, thereby by-passing the metabolic controls which normally exist in the animal; Correspondence to: F. Gannon, National Diagnostics Centre/BioResearch Ireland, University College,
Galway, Ireland. 0168-1656/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
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(c) The transfer of the DNA construct in vitro to a recently fertilized 1-cell ovum; (d) The reimplantation of the treated ovum into suitable recipient and (e) The analysis of the offspring. If the DNA construct has been integrated into the chromosome of the animal it is called a transgenic animal. Clearly the desired outcome from this protocol is the beneficial expression of the novel gene in the transgenic animal. As the injected DNA is integrated into the genome of the animal it should be transmitted in the germ line to the next generation. In this way very significant changes in the characteristics of animals may be achieved. Experiments of the type outlined above were first successful in mice (see below) and in recent years have been extended to other animals. This work is motivated by the need to: (a) Increase the efficiency of food conversion in domestic animals; (b) Generate animals with improved characteristics, and (c) Use animals as production units for compounds of therapeutic interest. In this review all three aspects will be considered as will the information obtained from laboratory animals which is pertinent to the development of transgenic commercial animals. The major question facing those who have recognised the potential of transgenics is whether or not they should be generated. This question will be addressed in the closing section of the review.
Transgenic mice Widespread public and scientific interest in transgenics was first aroused when the cover of Nature carried a photograph of two mice one of which was twice the size of its litter mate. The increased growth was a consequence of the insertion of a growth hormone gene into the pronucleus of a fertilized ovum (Palmiter et al., 1982). The generation of the 'super mouse' was the culmination of many years of effort in the relevant technologies. As is clear from the outline of the transgenic animal production method, a good understanding of how to manipulate embryos in vitro, how to microinject DNA with precision, how to make DNA constructs which can be expressed in animals, and how to reimplant ova into appropriately synchronised recipients were prerequisites for this dramatic demonstration of the technique. This particular experiment, while not the first successful attempt to make a transgenic animal (see for example Gordon et al., 1980; Constantini and Lacy, 1981; Gordon and Ruddle, 1981; Palmiter et al., 1982b) nonetheless indicated the awesome power of the technique. Some details of relevance to the generation of transgenic commercial animals will now be considered. Reviews which provide more detailed information on the topic of transgenic mice include those by Palmiter and Brinster (1985, 1986).
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Method of introduction of D N A into ova
A fertilised ovum is an intact and relatively impermeable entity. Methods had to be developed which would permit transfer of foreign D N A across this barrier. These methods are of two kinds; physical and biological. Currently the most successful approach is the physical microinjection of the D N A into the pronucleus of the ovum. Although requiring undeniable skill and specialist equipment, the large number of transgenic mice that have resulted from this method indicates that it is a reliable technique. For some time the only biological method available was the use of retroviruses as vectors of DNA. Embryos exposed to these vectors can become infected, allowing the DNA of interest to be integrated into the genome. However, this approach has several disadvantages: - The use of retroviruses is unlikely to be acceptable for the production of transgenic animals for human consumption; - Special containment facilities are required to ensure that safety norms are maintained; - Complicated host-vector combinations are a prerequisite to the use of retroviruses which are, of necessity, incapable of self replication; - When integration occurs, the single copy of the gene of interest that is inserted, is usually flanked by the long terminal repeats of the retrovirus which can interfere with expression; - Integration occurs when the embryo is already at a multi-cell stage of development with a consequent diminution of its effect on the founder animal and on the initial efficiency of germ line transfer. For these reasons retroviruses are not widely considered as a practical option in the long term for the delivery of DNA although they have been used successfully (for example Jaenisch, 1976; Soriano et al., 1986; Bosselman et al., 1989). Very recently a new biological alternative has been reported which combines efficiency with simplicity. In essence, the sperm of the animals is bathed in the D N A of interest and, by mechanisms that are not fully described, carries it into the ovum during in vitro fertilisation (Lavitrano et al., 1989). The efficiencies obtained for the generation of transgenic mice was 30% compared to 25% by the more complicated procedure of microinjection (Brinster et al., 1985). This new method is now under active consideration in other laboratories and if successful it is clear that it will be widely used for many different species.
Integration of foreign D N A into the g e n o m e
When the microinjection approach is used, several hundred copies of the D N A of interest are usually introduced into the pronucleus. Successful generation of transgenics demands that at least one of these be integrated into the genome. The mechanism by which this occurs is obscure at the molecular level. The data available show that the number of DNA copies which are intergrated and the sites of
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integration are variable. Usually several D N A copies are integrated in tandem in a head to tail fashion but other arrangements also occur (Palmiter et al., 1983). A hypothesis has been proposed whereby DNA integration occurs at random breaks in the chromosomal DNA (Brinster et al., 1985). These breaks are the rate-limiting step for integration. Interaction between the ends of the injected D N A and the chromosomal break points allows its insertion into the genome. This may explain the frequently observed lower efficiency of integration when using circular rather than linear DNA molecules (Brinster et al., 1985). The tandem array may then be formed either by homologous recombination between the inserted D N A and other injected DNA molecules, by ligation of the injected material in the pronucleus prior to integration or by amplification of the D N A when inserted into certain chromosomal locations. No evidence for any site-specific integration or of recombination between injected DNA and homologous mouse sequences has been found. Analysis of many integration sites may in time reveal more information on these points. However already, deletions, duplications and short novel sequences, found neither in the injected DNA nor in the area of integration, have been found at integration sites (Woychik et al., 1985).
Expression of transgenes Although the integration of foreign D N A in the genome can be demonstrated by techniques such as Southern blotting, a positive result from such analysis is academic if the inserted gene is not expressed. Our knowledge of the requirements for transcription and translation in eukaryotes is advancing rapidly and now includes roles for a variety of transcription factors which interact with the region 5' to the gene for maximum and controlled expression (see Dynan, 1989 for example). However, when transgenic mice were first produced it was unclear whether the mere provision of promoter and related sequences would be sufficient to allow expression of the gene. It was possible that: - The transgene would integrate in the 'incorrect' locus in the chromosome and this would override other influences in transcription; - The promoter region defined in vitro or in transient assays in cell culture might be inadequate in vivo; - The factors required for expression might be limiting; or - The hybrid gene might lack some subtle combination of sequences required for the efficient transcription of natural genes. Because of transgenic animal experiments, we now know the answers to many of these questions. The successful expression of sequences inserted into transgenic animals has shown that the incorrect chromosomal location does not necessarily prevent expression. However, it is frequently alluded to in explanations of the varying levels of expression obtained with different transgenics that contain the same DNA construct. The promoter regions used are frequently very short and they obviously are effective. Occasionally in vitro studies are not completely accurate in
159 their description of the requirements for transcription. One such example is the fact that sequences approximately 10 000 bases from the rat albumin promoter enhance expression in transgenics whereas such influence was not recorded during in vitro or in transfection studies (Pinkert et al., 1987). It is impossible to directly test for the adequate availability of transcription factors but one indication of a possible limitation is the fact that there are not normally correct gene dose effects in transgenic animals i.e. although a variable number of copies of the D N A are inserted into the genome, the product level in the animal does not relate to that number (see for example Palmiter et al., 1982a). This could be due to chromosomal location; however, when transgenic progeny are analysed, it appears that an alteration in the gene copy number does not always change the level of product. Because strong promoters are the usual choice for DNA constructs used in transgenics, it is possible that some factors required for efficient transcription are depleted when this extra demand is placed on the cell.
Tissue
specificity
of
expression
The mouse metallothionein promoter was used in the initial constructs which resulted in extremely high levels of growth hormone and large mice. It had been chosen because it is expressed in the liver and could be modulated by heavy metals and glucocorticoids. These controls were less than absolute in the transgenics and were of little practical importance. The control by zinc, however, did indicate that transgenic animals could maintain control features present in the promoter when it is not in its natural locus. An obvious extension of this was the demonstration that tissue specificity can also be achieved with constructs used in the generation of transgenics. Some promoters have a very restricted range of cell types in which they will function. Examples of these include elastase (Ornitz et al., 1985) and insulin (Bucchini et al., 1986) that are expressed in the pancreas or the crystallin gene (Overbeek et al., 1985) which is expressed in the eye. Transgenic mouse experiments (Swift et al., 1984; Seldon et al., 1986) showed that these and many other promoters act in an appropriate tissue-specific manner (Palmiter and Brinster, 1985). In some experiments, promoters from mammals other than mouse were used and tissue specificity was retained in the transgenic mice. A rapidly expanding literature now shows that the ability to express sequences in targeted cell types is used to: - Define promoter and cell specificity requirements by deletion and mutation experiments; - Examine the consequences of ablating certain cell types in animals (for example see Borrelli et al., 1989); - Describe the susceptibility of animals to expression of different oncogenes in different cells (for example see Brinster et al., 1984); - Assess the impact of increasing the circulating levels of certain hormones on the overall physiology of the animal.
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Inheritance From the design of the transgenic experiment which envisages the incorporation of DNA into the chromosome, it is clear that some of the offspring of transgenic founder animals should also be transgenic. It has been shown repeatedly that 50% of the offspring are transgenic, if the injected DNA integrates into a single chromosomal locus in the founder. Occasionally this does not occur. These cases are most readily explained by a degree of mosaicism in the founder animal (for example see Palmiter et al., 1984) which can occur if the injected DNA is not integrated into the chromosome prior to cell division. In microinjection experiments, with mice, the extent of mosaicism suggests that the DNA can persist in ova for up to three cell divisions. In addition some DNA combinations contain elements which allow for episomal persistence of the injected DNA, but this is not a frequent occurrence (Rassoulzadegan et al., 1986; Leopold et al., 1987). When the DNA fragments are initially inserted in the chromosome, multiple copies of the gene are frequently co-integrated (see above). One of the surprises from the early transgenic experiments was the alteration and editing of these sequences in the offspring. Different explanations have been proposed for this including a selection against the unusual structures which might result from multicopies or some influence on the locus due to inappropriate methylation. Once the editing has occurred in first progeny, the transgenic sequences then appear to be stable and are inherited like other autosomal genes. A final aspect of the inheritance of transgenes is the fact that transgenic animals are frequently less fertile than their non-transgenic litter mates. In this case the gene product and the cell type in which expression occurs are of importance. It has been suggested that a promoter which is active in germ cells diminishes the fertility of the animal. Also the presence of high circulating levels of growth hormone for example, perturbs the physiology of the animal thereby reducing its fertility. However, in most cases, transgenic mice with useful traits can be successfully used to establish colonies which perpetuate these characteristics.
Transgenic farm animals The results of transgenic mouse studies reinforce the idea that this model can be applied to animals of economic importance. The method and skills used to insert DNA into cell ova have been learned by many using the mouse system. The general requirements for D N A constructs which allow expression of genes of interest have been established and the ability to transmit these traits in the germline has been demonstrated. Nonetheless, the generation of transgenic farm animals presents technical difficulties which have not been encountered in laboratory species.
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Methodology of gene transfer in farm animals The choice of method for transfer of D N A to fertilized ova is based on the same reasoning as for mice and currently microinjection is the most widely used approach. This procedure in mice is aided by the fact that the ova are partly transparent and the pronuclei readily visualised. In species such as cattle and pigs, microinjection procedures are more complex because of the high lipid content and the opacity of the ova. This difficulty is now overcome by the use of centrifugation and the DIC (Nomarski) microscopy. Viable ova with discernible pronuclei can be obtained in this way. A second and major limitation when farm animals are used is the difficulty of obtaining large numbers of ova. The yield is increased by gonadotrophin administration but the average number of suitable ova obtained in cattle is typically about seven. The recovery of recently fertilised ova from cattle, sheep and other animals requires full scale surgery following artificial insemination. Then, following D N A microinjection, the ova are surgically transferred to the oviducts of synchronous recipient animals. The logistics and costs of the total procedure are major impediments to the production of transgenic farm animals. This can be further aggravated by the fact that the current expected frequency of production of transgenic calves is very low ranging from 0-2% of calves born from gene injected ova. Alternative methods for generating large numbers of ova required for efficient transgenic farm animal experiments include attempts to establish embryo carcinoma cell lines equivalent to those available for mice (Kuehn et al., 1987). These would be an unlimited source of starting material which could be regenerated into whole animals. An added attraction is the fact that selection of transgenic cells might be possible, by use of appropriate markers, prior to transfer to recipient animals. Another, more advanced development would be the use of in vitro fertilization and in vitro maturation procedures. These involve the use of preovulatory oocytes which can be matured and fertilized in vitro (Lu et al., 1987). Subsequent steps would involve microinjection of the foreign DNA, in vitro selection of ova that contain this D N A integrated into the genome and non-surgical transfer of these ova to recipient animals. Methods for identification of developing ova that have integrated the microinjected DNA will have to be very sensitive as development in vitro of some animal ova is blocked after a small number of cell division. The use of techniques such as the polymerase chain reaction (PCR) which amplifies defined D N A fragments approximately 1 million-fold may facilitate the detection of such ova (Saiki et al., 1988). This procedure therefore is likely to involve the removal of one or two biopsy cells from the developing embryo. These can then be analysed to confirm the presence and integration of injected DNA. The biopsied embryo should develop normally following such treatment as is evidenced by the ability of bisected embryo halves to resume normal development.
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Farm animals with improved characteristics As growth hormone (somatotropin) given in repeated injections to many animals can increase their efficiency of food conversion, growth characteristics and milk yield, it is not surprising that many of the transgenic experiments with farm animals have focussed initially on components in the growth endocrine cascade. The aim of these experiments is not to produce mammoth animals but to provide animals that are ready for slaughter at younger ages and have more effective food conversion. The production of animals with lower fat content, with larger litter sizes, with greater milk yields or with improved disease resistance are further targets for this research. In all of these instances the transgenic method is attempting to rapidly provide animals of a type which might be obtained in due course by the current standard agricultural methods. Work on transgenics can also be directed to altering animals in a way which is not possible by standard methods. It should be possible to change the content of milk by directing the expression of proteins of interest to the mammary gland. The resulting benefits could be nutritional or commercial depending on the protein involved. An example of the latter might be the increased expression of some caseins which might have useful applications when purified. An extension of this thinking would be to alter other biochemical characteristics in the animal. The current medical view is that animal fats are unhealthy. This is due to the presence in them of unsaturated fatty acids. A portion of these fatty acids could be rendered polyunsaturated by the inclusion in animals of the gene which codes for the enzyme delta-12-desaturase which is found in plants. It is estimated that approximately 30% of the animal fatty acids could be made polyunsaturated in this way (Gannon, 1986). An alternative use of transgenic farm animals is as bioreactors which produce therapeutic proteins of high value. Many of these proteins come from man or other mammals originally and are frequently modified by post-translational glycosylation or acetylation. When these genes are molecularly cloned, the choice of host in which they are expressed is frequently bacteria (which do not have the ability to glycosylate), yeasts (which modify proteins in a manner which does not exactly correspond to mammalian cells) or animal cells in bioreactors (Gannon, 1985) which are not yet cost-effective. Transgenic farm animals could be hosts of choice if the levels of expression obtained are economical. In addition, the transgenic animal may be uniquely suited for production of large or multi subunit proteins which need cofactors for their activity.
Expression in transgenic farm animals It is evident that transgenic farm animals could be of value in many different aspects of Biotechnology. One measure of this is the fact that currently over 40 applications are under consideration for transgenic animal patents (Ford, 1988; Fox, 1989). As a consequence, there are relatively few scientific publications which describe work in this area (Pursel et al., 1989).
163 The research reported relates to both animal improvement and protein production by transgenics in a number of species. In all cases a gene of interest must be expressed in a chosen tissue. The required end point has a major influence on the choice of promoter used in the DNA constructs. Typically, researchers involved with modification of the whole animal have selected promoters which function well in the liver as it is the largest internal organ and its products are readily transferred to the blood stream. The mouse metallothionein promoter fulfulls that requirement but also expresses in a wide range of other cell types. This can be an advantage if the product is thought to act as an autocrine but may be a disadvantage if its over-expression disrupts the normal function of sensitive cells. Alternatives to the use of the metallothionein promoter are liver-specific promoters. In this context we have recently isolated the bovine serum albumin promoter for use in transgenic studies (S. Power, T. Barry, and F. Gannon, unpublished). An efficient liver promoter could also be of use where therapeutic products are required because protein purification from blood can be readily performed. However, many of these products could have detrimental effects on the animal (for example over-production and circulation of blood clotting factors or lymphokines). For this reason, examples quoted below will show that promoters which are specific to the mammary gland, from which the product is secreted in milk, have been the choice of most groups who wish to use the animals as bioreactors.
Examples of transgenic farm animals Following the successful generation of the super mouse it was clear that the mouse metallothionein promoter-human or rat growth hormone D N A construct would be used in other species. The first published results following that came from Hammer et al. (1985) using rabbits, sheep and pigs. The results were disappointing (Lovell-Badge, 1985) and showed clearly that the application of transgenic technology to farm animals would not be simple. Low levels of expressed growth hormone were detected in rabbits and pigs but had no effect on growth rate. Numerous attempts have since been made to generate transgenic farm animals and these have been most successful with sheep and pigs. Interestingly the focus of work with sheep has been to use them for expression of high value proteins whereas pigs have been targeted for modification of the whole animal.
Sheep The work with transgenic sheep recently culminated in the expression in the mammary gland of the human blood clotting protein, Factor IX (Clark et al., 1989). The sheep ~-lactoglobulin promoter which was previously shown to be specific for the mammary gland and to express factor IX in mice (Simons et al., 1987) was used. The overall efficiency of the experiment in sheep was approximately 2% illustrating the need for large numbers of animals when embarking on such studies (Clark et al., 1987; Simons et al., 1988). The levels of expression of Factor IX or human
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al-antitrypsin in these experiments were low and cannot yet compete with other expression systems. Work is continuing to optimise the levels of production. There have also been reports on the development of transgenic sheep to over-express epidermal growth factor and other proteins of value. Many of these have not been published in a complete form and are therefore difficult to comment upon. Nonetheless, the impression is created that a number of research groups, usually with commercial collaboration, have succeeded in establishing the technology to generate transgenic sheep.
Pigs In a manner analogous to sheep, a number of groups have reported success in generating transgenic pigs. These have focussed primarily on the over-production of human growth hormone. Brem et al. (1985) were among the first to report the birth of a transgenic pig, after the transfer of 268 zygotes, again underlining the practical difficulties of performing these experiments. Since then, constructs using either the Moloney MuLV promoter (Ebert et al., 1988) or the mouse metallothionein promoter (Vize et al., 1988) have resulted in the over-expression of growth hormone in a small number of pigs, none of which were healthy. A very recent detailed description (Pursel et al., 1989) of a series of experiments with transgenic pigs in which bovine growth hormone levels are elevated showed that individual pigs had increased weight gain but the authors suggest that this should not be over-interpreted because individual controls could also differ from the mean by as much as 30%. When the offspring from two transgenic lines of pigs were analyzed, it was shown that in one case transgenic pigs grew 15% faster than littermate controls if the diet was supplemented with extra protein and lysine. In addition, significant improvements in feed efficiency and a decrease in carcass fat were noted. However, a number of pathologies in the pigs were also described by the authors who believe that these could be diminished by a combination of careful selection of the founder animal and by limiting transgene expression to the rapid growth phase of the animals (Pursel et al., 1989), A number of research projects are suggested by this study including the description of systems that can be controlled by exogenous factors, a complete characterisation of the factors which interact to result in promoting growth, the influence of nutrition on defining an optimal circulating concentration of growth hormones and the consequences of the use of promoters other than metallothionein.
Cattle Many groups are working towards production of transgenic cattle with improved characteristics. Again the published literature provides little in the way of detail. Transgenic calves with improved growth have not been formally reported but reference has been made, without documented data, to calves which express antigenic epitopes of virus (Church, 1987) and to the expression of fl-interferon (see Van Brunt, 1988). The effects on the animals are unknown. This paucity of information contrasts with the major commercial interest in this species and is
165 reflected in background information articles in biotechnology journals (e.g. Klausner, 1988; Van Brunt, 1988) in which ongoing experiments using actin or B-casein as promoters for the expression of the estrogen receptor or growth factors were referred to. Presumably, when patent aspects are clarified, this information will become available. Rabbits
Transgenic rabbits have also been produced. Because rabbit ova are transparent, microinjection is less complicated than for other species. Some basic scientific experiments have been extended from transgenic mice to transgenic rabbits (Knight et al., 1988). Growth hormone has also been expressed in transgenic rabbits (Hammer et al., 1985) and proteins of commercial importance have been produced in rabbit milk (L.M. Houdebine, personal communications). It would appear that the latter is the application which might be of relevance in the future for transgenic animals of this species. Fish and chickens
Although microinjection into the pronucleus is the method of choice for most animals, it is not appropriate for either fish or chickens. For chickens it would appear that retroviruses might provide the best approach given the physical difficulties of working with avian eggs. Salter et al. (1987) and Bosselman et al. (1989) have succeeded in transferring proviral fragments to chickens and demonstrated mendelian inheritance of this DNA. Similar results using drug resistance gene markers have been reported by Nigon (1988). These results clearly point the direction in which research on transgenic chickens will proceed. The challenge to apply transgenic methods to fish which are grown commercially is at first sight simplified by an unlimited supply of eggs, easy in vitro fertilisation and development ex utero. A variation of the microinjection scheme is used with the DNA delivered to the cytoplasm rather than the nucleus (Chourrout et al., 1986). For some noncommercial species microinjection into the nucleus is possible and has been successful (see review McLean et al., 1987). A difficulty in reviewing the literature on transgenic fish relates to the persistence and indeed amplification of episomal injected DNA. To date there are no detailed descriptions of transgenic fish that express the transferred genes (see Powell et al., in press). Inheritance of the foreign DNA has been demonstrated (Guyomard et al., 1989) and occasional reports of expression of foreign genes several weeks after microinjection (Ozato et al., 1986; McEvoy et al., 1988) have appeared but the lack of familiarity with the molecular biology of fish is proving to be a major limitation in the preparation of DNA constructs which will be effective. The recent cloning of the salmon growth hormone gene (Johansen et al., 1989; Lorens et al., 1989) and of a variety of other fish genes (Wolff and Gannon, 1989; R. Powell, L. Byrnes and F. Gannon, unpublished) should provide a more solid basis for these studies in the near future.
166 The future
In a very short period of time transgenics has moved from a futuristic concept to a challenging reality. Man can now alter the genetic characteristics of m a n y animals. At a technical level, work is forging ahead to improve and extend these techniques. Methods to increase the supply of ova, to facilitate the transfer of the D N A constructs to the ova, to establish selection procedures for transgenic ova prior to reimplantation, and to increase the control, specificity and level of expression of selected genes are all under active study. With time it is clear that species for which current methods are inefficient will be added to the repertoire available to scientists. Already much has been learned from transgenic studies which has advanced our understanding of the normal and the pathological in mammals and this will continue. New whole animal test systems have become available to allow harmful effects of compounds to be identified (Fox, 1988). Early examples show that farm animals can be used in the manufacture of complex products of therapeutic benefit. As different construct and animal combinations are prepared it will be possible to make rational commercial decisions on the future of this technology. Finally, animals, fish and birds have been generated which can be considered to be the first in a series which should eventually yield species with improved or altered characteristics. The commercial benefits from this are potentially large. The advent of a potent new technique with clear commercial potential would normally give rise to extravagant extrapolations of the value of the development. This is not happening for transgenics and indeed one analysis of the current climate is that there will be very restricted use of transgenics. There are m a n y converging reasons for this ranging from concern for animal welfare to commercial doubts. When a fragment of D N A is inserted into the genome of an animal the precise outcome is unknown. Harmful mutations can occur, although these are rare. Greater problems are encountered in the context of the altered physiology of the animal due to the over-expression of a potent biochemical. It is now documented that some animals are prone to a variety of ailments when they are transgenics. This is best documented for pigs with increased growth hormone levels (Pursel et al., 1989). It seems reasonable that combinations of D N A constructs and animal will be obtained for many species in which the benefits are not diminished by the side effects. In the meantime want on and unnecessary hardships to animals must be avoided. Any transgenic offspring that have disabilities must be quickly and humanely killed. Concern for animal welfare and mere pragmatism, which recognises that such animals could not be the basis for a commercial venture, demand this. If transgenic farm animals with useful characteristics are made, then a number of other questions must be faced. The practice of using constructs which were shown to be useful in mice for experiments with larger animals still results in the incorporation of D N A fragments from heterologous species or from viruses. It is not helpful for the image of transgenics and, for some, of the ethics of the experiment if the key component originated from man, mouse, rat or viral DNA. For this reason we, and others, have sought homologous promoters and active genes for the purpose of making transgenic farm animals. This trend is certain to continue in the future.
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A further question relates to the free release of recombinant organisms. For m a n y domestic animals the 'release' can be easily controlled. When any p r o g r a m m e moves to the next phase in which the animals are used for breeding purposes, the long-term consequences will have to be adjudicated upon. Those who place the environment at the top of a list of priorities will demand, as a minimum, that great caution be exercised. Given the scientific impossibility to exclude absolutely any negative consequences of free release, it would seem likely that a number of generations of transgenics resulting from diverse cross-breeding experiments will be required for study in well-controlled environments. The resolution of many of the practical problems related to transgenics can be achieved as outlined above. In biotechnology, the step from an ability to make a product to the use of this ability is decided by consideration of commercial criteria. The uncertainty related to transgenics, because of the environental and animal welfare aspects, has a very negative influence on businesses which might become involved in transgenics. The benefits of animals with improved growth rates, food conversion or decreased fat content can be appreciated by those industries, but they have not been presented with a clear example of a healthy transgenic animal with these traits. When this happens the analysis by m a n y companies of the future situation will undoubtedly become more positive. Without industrial investment there is the possibility that the research in transgenics will lose m o m e n t u m and become a laboratory-based technique. National and international funding agencies are currently responding to the anti-transgenic lobby in a way which makes it difficult to promote more research in this area. If transgenics becomes, in time, an example of regulatory consideration impeding a controlled and informed development of a technique, a number of direct benefits will be lost. The therapeutic products from biotechnology which carry the hopes for treatment of many human diseases may in time be most appropriately obtained by expression in transgenic animals. The testing of new products for toxins may in fact be best performed on whole animals that are sensitized to identify the carcinogens rather than in bacteria or in cell culture where only a limited number of cell types and sets of conditions can be tested. The accepted justification for decades of breeding programmes has been to make animals that either produced more milk or meat in a cost-effective manner. This aim of work on transgenics is equally laudable and the prospects for significant progress towards these goals in an accelerated time frame are real. It would seem an error if the balance of scientific potential and of regulatory prudence did not find an agreed basis for further progress in the important area of transgenics. If such a basis is not found then one must wonder which will be the next aspect of science to be obstructed.
Acknowledgements Work referred to in this review includes aspects funded in the laboratories of F. G a n n o n and J. Sreenan by the E.C. Biotechnology Action Programme.
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