Transgenic swine for biomedicine and agriculture

Theriogenology 59 (2003) 115±123

Transgenic swine for biomedicine and agriculture R.S. Prathera,*, R.J. Hawleyb, D.B. Carterc, L. Laia, J.L. Greensteinb a

Department of Animal Sciences, University of MissouriÐColumbia, 920 East Campus Drive, Columbia, MO 65211-5300, USA b Immerge BioTherapeutics, Charlestown, MA 02129, USA c Department of Veterinary Pathobiology, University of MissouriÐColumbia, 920 East Campus Drive, Columbia, MO 65211-5300, USA

Abstract Initial technologies for creating transgenic swine only permitted random integration of the construct. However, by combining the technology for homologous recombination in fetal somatic cells with that of nuclear transfer (NT), it is now possible to create speci®c modi®cations to the swine genome. The ®rst such example is that of knocking out a gene that is responsible for hyperacute rejection (HAR) when organs from swine are transferred to primates. Because swine are widely used as models of human diseases, there are opportunities for genetic modi®cation to alter these models or to create additional models of human disease. Unfortunately, some of the offspring resulting from NT have abnormal phenotypes. However, it appears that these abnormal phenotypes are a result of epigenetic modi®cations and, thus, are not transmitted to the offspring of the clones. Although the technique of producing animals with speci®c genetic modi®cations by NT has been achieved, improvements to the NT technique as well as improvements in the culture conditions for somatic cells and the techniques for genetic modi®cation are still needed. # 2002 Published by Elsevier Science Inc. Keywords: Transgenic; Nuclear transfer; Cloning; Xenotransplantation

1. Introduction Technologies for germline transmission of transgenes in swine ®rst included only pronuclear injection [1], but later evolved to include sperm-mediated transfection [2], oocyte transduction [3] (also referred to as transgametic gene transfer [4]), and NTmediated transgenic pig production [5]. More recently, a speci®c genetic modi®cation was made to the a(1,3)-galactosyltransferase (GalT) gene prior to creation of the pigs by NT [6,7]. Each of these technologies has certain advantages and disadvantages that include * Corresponding author. Tel.: ‡1-573-882-6414; fax: ‡1-573-882-6827. E-mail address: [email protected] (R.S. Prather).

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ease of making the desired genetic modi®cation, the type of modi®cation that can be made, and the stability of the transgene in subsequent generations. With pronuclear injection, sperm-mediated transfection, and oocyte transduction, DNA constructs are added with little control over the site of integration or the number of copies that integrate. Oocyte transduction is further limited by the size of the construct that can be inserted. Thus, the regions ¯anking the site of integration and, to some degree, the untranslated regions included with the construct control transcription of the transgene. In contrast, by using homologous recombination technology in combination with NT, a more speci®c modi®cation can be made in cells in culture prior to the NT [7]. Central to each of these technologies is the ability to culture pig embryos in vitro. This topic has been previously reviewed at these meetings [8,9]. The importance of the development and identi®cation of de®ned, and thus repeatable, in vitro conditions for oocyte maturation, fertilization, and subsequent culture in the pig should not be underestimated. With micro-injection of in vivo-produced pronuclear stage embryos and immediate embryo transfer, the culture time can be minimized. However, if the goal is to produce embryos in vitro and perform a non-surgical embryo transfer at the blastocyst stage, then optimized culture conditions are even more imperative. Transgenic pigs have been produced by using in vitro technology [3]. Not only were these the ®rst transgenic pigs resulting from in vitro embryo production, but one was also the ®rst piglet from in vitroproduced embryos cultured to the blastocyst stage prior to embryo transfer, and it was the ®rst example of oocyte transduction in the pig [3]. These gilts express eGFP in various tissues, as do their offspring. Transgenic pigs with random insertions have proven very valuable for biomedical research, and they provide a great potential for agriculture. It is anticipated that the ability to perform speci®c genetic modi®cations will have enormous utility in these ®elds. More speci®c genetic modi®cations via application of homologous recombination technologies and NT to the production of transgenic pigs are not straightforward. Speci®cs of biomedical and agriculture applications will be discussed subsequently, as well as strategies to perform homologous recombination ef®ciently and to care for animals that may have an abnormal phenotype as a result of the NT technology. 2. Biomedical applications of speci®c genetic modi®cations Swine have been used in biomedical applications for many decades as a model for human disease processes, as a genetically de®ned model for surgery and transplantation, and as a source of human therapeutics. All of these uses could be enhanced by the ability to speci®cally and non-speci®cally modify the pig genome. The majority of work to date in dominant transgenesis by micro-injection has been directed at optimizing pigs to serve as a donor of cells, tissues, and organs for transplant into humans (xenotransplantation). Clinical use of transplantation has become one of the major treatments for many diseases associated with terminal organ failure. This success has created the secondary issue: lack of human organ supply, which has greatly limited the number of patients who can receive such life-saving treatment. Currently (http://www.unos.org/ Framedefault.asp?CategoryˆNewsdata), over 80,000 patients in the United States alone

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are waiting for organs, yet only 24,000 transplantations were performed in 2001. Many programs have been initiated to increase donor awareness, and, in some countries, a presumed consent law has been put into practice. Although these efforts have increased the donation rate, it has been estimated that none of these efforts can adequately address the shortage of human donor organs worldwide [10]. Xenotransplantation has the potential to offer a practical solution. Perhaps surprisingly, the pig is thought to be the most suitable non-human organ source. The physiology of most of the major organs is similar in pigs and humans; pigs can be easily bred, they give birth to large litters, their gestation time is relatively short; their use as donors of organs can be ethically balanced by their general use as a food source (over 95 million pigs are used for food per year in the United States alone); the size of their organs can match clinical requirements, and they can be housed in a manner to decrease the potential infectious disease concerns. One of the major constraints to using pig organs for xenotransplantation is human natural antibody-mediated HAR of pig tissues. Humans and old world monkeys possess natural anti-pig antibodies that are speci®c for a(1,3)-galactosyl epitopes on pig cells. Pigs (and most other species) express a(1,3)-GalT that causes a terminal galactose a(1,3)-galactose modi®cation to be present on essentially all glycoproteins. Hyperacute rejection is triggered by ®xation of a recipient's preformed natural antibodies on graft endothelium, and subsequent activation of the complement cascade, which leads to formation of extensive thrombosis within the graft and its necrosis within minutes after grafting. Previous approaches to overcoming HAR in pig-to-primate xenotransplantation have included expression of human regulators of complement activity in transgenic pigs, depletion of recipient complement activity, depletion of speci®c natural antibody, and inhibition of recipient antibody production [11]. In all cases, these approaches have proven, alone or in combination, to eliminate or delay HAR, but appear to be either insuf®cient to eliminate critical organ damage or to have only transient effectiveness [11]. Carbohydrate remodeling has been recently attempted by using two different enzymatic approaches: the transgenic addition of a fucosyltransferase [12], which is capable of competing for terminal sugar modi®cation, or a glucosaminyltransferase [13], which is capable of altering sugar biosynthesis at an earlier point in the pathway. Neither approach completely eliminates the expression of galactose a(1,3)-galactose epitopes, and it has been estimated that removal of 95% of these epitopes would not be suf®cient to block the biological effects of the speci®c natural antibody [14]. Interestingly, long-term study of families bred from these transgenic founders has illustrated some of the dif®culties with random transgenesis and pharmaceutical uses of these pigs. Concatamers of the inserted genes may be found in the genome, gene diminution may occur in offspring, and the expression levels may vary in the offspring. This can present a dif®culty when developing a herd that requires quality assurance and control at the regulatory level. The ability to speci®cally target genes via homologous recombination may simplify some of these issues. Elimination of a(1,3)-GalT from the pig is an innovative approach to the problem of HAR and delayed vascular rejection, as both processes have been associated with natural antibody. We have shown that loss of a(1,3)-GalT activity, through gene-targeted interruption, eliminates or greatly reduces complement-mediated lysis of pig endothelial cells by both human and non-human primate sera in vitro (Hawley, unpublished data); thus, this

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strategy has great potential utility in controlling the immune response to a porcine xenotransplant. We had the goal of producing a GalT knockout pig for many years and evaluated many technologies to achieve this goal. Until both NT cloning in pigs and homologous recombination targeting was achievable, this had not been practical. Putting these technologies together has allowed us to produce a pig with one copy of the GalT gene removed [7]. We anticipate that the removal of the second copy of the gene, by either breeding or retargeting, will allow us to evaluate the ability of these organs to bypass the antibody-mediated rejection of pig organs in non-human primates and will serve as a platform for generation of products for human xenotransplantation. Making speci®c genetic modi®cations in the pig opens the possibility of producing recombinant products in animals for biomedical or nutraceutical uses and the possibility of producing disease models for research and drug development. Goats and cows are already being used for recombinant protein production. The pig offers the advantage of shorter gestation time, shorter time to sexual maturity, and production of larger litters. The cost effectiveness of using the pig may become the driving force in the decision of which species to choose. In areas of biomedical development, where the pig is already a product source, for example heart valves or skin, it may be possible to add or remove speci®c genes that would bene®t the medical use that is currently being served. The pig is already established as a model for human physiology and pharmacology. Many of these models could be made more relevant to the development of human therapeutics by the addition of the human gene product that is being targeted. It is also possible to utilize the ability to manipulate the pig genetically to produce models of human genetic disease. For example, it is now known that the structure of the cystic ®brosis transmembrane conductance regulator is mutated in cystic ®brosis [15]. Therefore, it is simple to propose that the expression of this mutated receptor in a transgenic pig would allow basic research and drug development to occur in an expedited fashion. As our understanding of the human genome increases, the possibilities are unlimited. The only issue is the cost of developing the transgenic animal and the time to breed the resulting modi®ed animals following the NT cloning. As the ef®ciency of the techniques increase, the ability to produce these animals will increase, and the costs will decrease. This work will impact the future of biomedicine in a positive manner. 3. Agricultural applications of genetic modi®cations As reviewed over a year ago [16], genetic modi®cation in swine could have many agricultural applications. Changes in the genome could (1) alter the carcass composition such that it is a healthier product, (2) produce pork faster or more ef®ciently, (3) create animals that are resistant to speci®c diseases, (4) reduce the major losses normally observed during the ®rst month of swine embryogenesis, and (5) create animals that are more environmentally friendly. Some examples that are already in existence include animals that contain exogenous growth hormone [17] and insulin-like growth factor-I [18] genes that may impact the ef®ciency of production as well as the quality of the product. In the same vein, Bleck et al. [19] generated sows that produced bovine a-lactalbumin in their milk. The piglets that

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nursed these transgenic sows had increased growth rates (cited in [16]). One of the major problems associated with con®nement rearing of swine is the volume and disposition of animal waste. Because pigs cannot digest the phosphorus in phytate, large amounts of phosphorus are excreted in the manure. Golovan et al. [20] introduced a phytase gene that is expressed in the salivary glands of the pigs. These animals produce manure with less phosphorous. Another important area may be the creation of animals that produce antibodies in their milk that would make those animals that nursed passively resistant to diseases such as transmissible gastroenteritis [21]. Genetic changes requiring modi®cation to a speci®c gene include myostatin. Myostatin has a naturally occurring mutation in cattle that results in additional muscle growth. Creating such a mutation in the pig may result in animals that grow more ef®ciently or have a better quality product [22]. Future applications of the production of transgenic pigs for agriculture are only limited by our understanding of the biological system and our imagination. 4. Strategies for homologous recombination in swine Until recently, speci®c modi®cation of mammalian genomes has been limited to certain strains of mice, where gene-targeting technology could be applied in embryonic stem (ES) cells and modi®cations introduced into the genome through chimeric blastocyst production. The same ends can now be achieved in other species through NT with speci®cally modi®ed somatic cells, but the lifespan of cultured somatic cells places constraints on vector design, screening strategies, and the degree to which potential donor cell lines can be assessed prior to NT. Furthermore, the current inef®ciency and cost associated with production of animals by NT dictates that donor cell lines be as near clonal as possible. Because the rate of targeted clone generation with standard gene targeting vectors is typically of the order of 10 6 or less, expression of a selectable marker gene from the modi®ed locus is required unless the desired genetic modi®cation itself produces a selectable phenotype. Published reports of targeted gene modi®cation of pigs [6,7] and sheep [23,24] have used fetal ®broblastic cell lines and gene-trap vectors, in which expression of a drug-resistance marker is dependent upon insertion into an actively transcribed locus. Initially developed as a means to decrease the number of non-targeted, transformed clones that need to be screened, such vectors also have the advantage of decreasing the time required for drug selection, as transiently transfected cells do not express the resistance marker. Even with such vectors and optimally designed selection strategies, a considerable proportion of potentially targeted cell clones will senesce before selection, expansion, and characterization can be completed [6,7,23,24]. Nonetheless, a good proportion of loci of interest can potentially be targeted using similar methodology. Gene-trap vectors cannot be used for a knock-in with an exogenous promoter or modi®cation of loci that are not expressed in fetal ®broblastic cells; targeted modi®cation of such genes presents additional challenges. In some limited cases, it may be possible to establish alternative somatic cell lines that express the gene of interest and have the proliferative potential necessary for selection of modi®ed donor lines. More generally, targeting vectors can include a marker gene with a ubiquitously expressed exogenous

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promoter. Such vectors may require transfectant selection for longer periods or at a much reduced cell density and will always require a considerably expanded screening effort. For loci with low targeting rates, this approach may not be practical. Recently, a number of approaches for introduction of short sequence alterations using small DNA fragments or RNA/DNA duplexes have been developed for gene therapy applications [25]. In some cases, the rates of sequence alteration may be high enough to permit isolation of genetically modi®ed donor lines by single-cell cloning directly from transfected somatic cells. Isolation of readily transfectable and selectable cells with high proliferative potential and long-term karyotypical normalcy, similar to murine ES and EG cell lines, has the potential to overcome the limitations currently encountered with somatic donor cell lines. Primordial germ cell-derived lines have been isolated from pig fetuses, and transfected lines have been shown to contribute to chimera formation when injected into pig blastocysts [22,26]. Although germ line transmission has not been demonstrated with non-murine lines, such cells may be well suited to genetic modi®cation via NT. We may anticipate that similar lines will greatly expand the ease and range of genetic modi®cations that can be introduced into large animal genomes by NT in the near future. 5. Implications of abnormal phenotypes in animals derived from NT Although there are gestational losses in NT pregnancies, our focus will be on parturition and the offspring. There are many reports of dams that are carrying NT pregnancies that show few signs of impending parturition (sows [27], ewes [28] and cows [29,30]). When offspring are produced either by natural parturition or caesarean, some offspring exhibit respiratory distress [27,31,32]. Administration of pulmonary surfactant has been used to attempt to reduce the symptoms of respiratory problems in both cattle and pigs [27,29]. Many groups have reported sudden or unexplained death of cloned pigs, lambs, and calves. Other reported abnormalities include contracted tendons and cardiovascular abnormalities [7,27,29,33]. Some abnormalities appear to be species-speci®c, as cloned piglets and mice appear to have normal birth weights [27,34,35], whereas cloned calves and lambs have large birth weights [32,34,36]. There is some suggestion that immune function may be compromised in cattle [37], but cloned pigs appear to respond to vaccination [27]. In cattle, some of these abnormal phenotypes may be associated with the donor cell type, as in one study cells from adults resulted in more abnormalities [38]. Many of these phenotypic abnormalities can be managed, e.g. parturition can be induced, mammogenesis can be enhanced, supplemental oxygen and surfactant can be administered to offspring, and contracted tendons can be splinted [27]. Although some groups have reported abnormal phenotypes resulting from NT in swine, others have seen few problems [39±42]. However, it should be noted that at least some of these abnormal phenotypes are not passed on to offspring. This includes both the large birth weights observed in cattle [43] and obesity in mice [44]. Our ®rst transgenic pig (402-2) produced by oocyte transduction [3] was the result of IVM, IVF, and culture to the blastocyst stage prior to embryo transfer. This gilt had a ¯exor tendon contracture similar to that seen in NT and in vitro-derived animals. Only one of her four clones had a ¯exure tendon contracture [5], and none of her 24 offspring have had this phenotype (Prather et al., unpublished). The cause of the abnormal

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phenotypes is likely epigenetic and at least partially controlled by DNA methylation [45± 47]. Thus, these abnormalities are a management concern during the ®rst generation. If the animals can reach sexual maturity and reproduce, i.e. transmit the desired gene, then the genetic modi®cation can be successfully introduced into the species of choice. 6. Summary Nuclear transfer technology in combination with strategies to target speci®c genes has resulted in gene-targeted swine. Now, many speci®c genetic modi®cations in swine can be contemplated that will have rami®cations in both production agriculture and human medicine. Improvements are still needed in the basic technique of NT, the conditions for culturing cells, and the method of genetic modi®cation. Because ES cells in swine have not been demonstrated, an embryonal carcinoma or primordial germ cell line that has more proliferative capacity may provide more utility than fetal cells. Improved methods in making the genetic modi®cation are also needed so that large regions of a chromosome can be changed without concern for isogenicity. Finally, development and application of the technologies for creating speci®c genetic modi®cations is dependent upon in vitro conditions for maturing and fertilizing oocytes and culturing embryos. Although these conditions are compatible with normal development to term, further re®nements are needed. Acknowledgements Work presented in this review was supported by NIH grants R44 RR15198 and R01 RR13438. References [1] Hammer RE, Pursel VG, Rexroad C, Wall RJ, Bolt DJ, Ebert KM, et al. Production of transgenic rabbits, sheep and pigs by microinjection. Nature 1985;315:680±3. [2] Sperandio S, Lulli V, Bacci ML, Forni M, Maione B, Spadafora C, et al. Sperm-mediated DNA transfer in bovine and swine species. Anim Biotechnol 1996;7:59±77. [3] Cabot RA, Kuhholzer B, Chan AWS, Lai L, Park KW, Chong KY, et al. Transgenic pigs produced using in vitro matured oocytes infected with a retroviral vector. Anim Biotechnol 2001;12:205±14. [4] Chan AWS, Homan EJ, Ballou LU, Burns JC, Bremel RD. Transgenic cattle produced by reversetranscribed gene transfer in oocytes. Proc Natl Acad Sci USA 1998;95:14028±33. [5] Park KW, Cheong HT, Lai LX, Im GS, Kuhholzer B, Bonk A, et al. Production of nuclear transfer-derived swine that express the enhanced green ¯uorescent protein. Anim Biotechnol 2001;12:173±81. [6] Dai YF, Vaught TD, Boone J, Chen SH, Phelps CJ, Ball S, et al. Targeted disruption of the alpha 1,3galactosyltransferase gene in cloned pigs. Nat Biotechnol 2002;20:251±5. [7] Lai LX, Kolber-Simonds D, Park KW, Cheong HT, Greenstein JL, Im GS, et al. Production of alpha-1,3galactosyltransferase knockout pigs by nuclear transfer cloning. Science 2002;295:1089±92. [8] Abeydeera LR. In vitro production of embryos in swine. Theriogenology 2002;57:257±73. [9] Prather RS, Day BN. Practical considerations for the in vitro production of pig embryos. Theriogenology 1998;49:23±32.

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