Transgenic pigs expressing plant genes

Fatty acids are a group of lipids that consist of a carboxyl group plus a long hydrocarbon chain. Saturated fatty acids have single carbon-carbon bonds, whereas unsaturated fatty acids contain one or more double bonds within the hydrocarbon chain. Polyunsaturated fatty acids (PUFAs) have two main functions: the first is a critical role in membrane structure where they confer fluidity and flexibility, and the second is their role as precursors of prostaglandins, leukotrienes, and glycolipids, all of which are essential in the regulation of various biological functions. Mammals lack the enzyme to create double bonds between the ninth carbon (from the carboxyl end) and the terminal methyl group and thus cannot synthesize linoleic acid (18:2n-6) and α-linolenic acid (18:3n-3), making these fatty acids an essential component of the mammalian diet (vitamin F). Plants, including spinach, produce these essential PUFAs by desaturating oleic acid (18:1n-9) at positions C-12 and C-15 to produce 18:2n-6 (linoleic acid) and 18:3n-3 (linolenic acid). Thus, the fatty acid desaturases constitute an important category of enzymes involved in the synthesis of PUFAs. The genes for the desaturases have been identified in various plant systems and in simple organisms including Caenorhabditis elegans (1). These enzymes have also been shown to be functional in mammalian cells on transfection (2). The article by Saeki et al. (3) in a recent issue of PNAS takes this research a significant step further by showing constitutive expression and activity of the Δ12 spinach desaturase gene (FAD2) in transgenic pigs. Levels of linoleic acid (18:2n-6) in adipocytes collected from transgenic pigs and cultured in vitro were ≈10-fold higher than in wild-type control cells. Biopsied white adipose tissue from transgenic pigs contained ≈20% more linoleic acid than that of the nontransgenic controls. This study is the first time that a plant gene has been functionally expressed in a complex mammalian system. This success may open the road toward the production of pork that is better for the consumer. Both cardioprotective effects and anticancer activity have been documented for the n-3 fatty acids, which are also important for normal function and development of the retina and the brain. For these reasons, a large market has developed for fish-oil capsules consumed by health-conscious individuals. Pigs have been fed a PUFA-rich diet, which has improved processing and increased PUFA concentrations in pork. The article by Saeki et al. is important for its interesting approach to the production of more healthful pork and diversification of the range of products available from domesticated animals.

In the study by Saeki et al. (3) microinjection technology was used to create FAD2-transgenic pigs. This well established technology involves injection of several thousand copies of a particular gene into the pronuclei of zygotes, a developmental stage shortly after fertilization when the sperm tail has vanished and the sperm head is transformed into the male pronucleus, soon followed by syngamy and the concomitant recombination of the paternal and maternal genome. This procedure has been successfully used for the production of transgenic animals for almost 20 years since its first successful demonstration in 1985 (4). The procedure works reliably but has several significant drawbacks (5). It is highly inefficient as judged by the low yield of transgenic off-spring, the random integration of the foreign gene into the host genome, and the resultant variable expression pattern of the transgene. Furthermore, the technology only allows addition of a foreign gene as opposed to deletion of an undesirable gene. The microinjection approach does not permit prescreening of the embryos in vitro for the presence of the foreign gene before transfer to the recipients. These limitations of the technology require screening after birth, which makes the whole approach extremely labor- and cost-intensive (5). A striking observation by Saeki et al. is the high degree of mortality in founders and the F₁ generation. Because of the random integration process, the transgene can insert in and damage any active gene locus (insertional mutagenesis). An alternative explanation for the high mortality could be the significant alteration in the embryonic lipid profile caused by the transgene. The porcine embryo is unique in its high intracellular lipid content, which is associated with its sensitivity against freezing or in vitro production (6).

Saeki et al. present, for the first time, a plant gene expressed in a complex mammalian system.

Saeki et al. (3) observed that levels of unsaturated fatty acids were >10-fold higher in preadipocytes derived from adipose tissue of transgenic pigs and cultured in vitro than in intact fat tissue from transgenic pigs without cultivation. Only one of the two transgenic pig lines had PUFA levels significantly higher than nontransgenic controls. Transgenic expression of a specific gene in a complex mammalian system usually does not mimic that observed under simple in vitro culture conditions. In addition, the FAD2 gene construct was based on a cDNA (3). Early work in transgenesis demonstrated that cDNAs are frequently expressed poorly in transgenic offspring (7, 8), because introns can contain enhancer elements that increase the transcriptional activity of homologous or heterologous promoter elements (9). Introns can also contain sequences that may facilitate transcription by affecting nucleosome composition and higher-order packing of DNA within the chromosome (10). Previous work has shown that the low expression of cDNA constructs can at least partly be overcome by adding homologous or even heterologous introns to the construct (7, 8). An improved construct, based on a genomic clone of the FAD2 gene, could produce a more significant effect on fatty acid composition under in vivo conditions. The adipocyte P2 promoter regulates the expression of one specific member of the intracellular lipid-binding protein family. A ≈540-bp enhancer element that is thought to be located at kilobase -5.4 is critical for high expression from this promoter (11). The fact that this promoter is highly specific for adipocytes is confirmed by Saeki et al., because they did not observe any significant ectopic expression of the transgene.

The work of Saeki et al. (3) is part of an international effort to improve pig production by transgenesis. Transgenic pigs have been created for several purposes (5). The first generation of transgenic pigs expressing human growth hormone (GH) showed dramatic effects in growth rates, feed conversion, and body composition, but it exhibited serious pathological side effects that were attributable to the high level of GH expression (12). In the eyes of the public, this was erroneously associated with the technology of transgenesis itself. Subsequently, GH transgenic pigs were produced that show the desired economically important traits without pathological side effects (13). In the context of the results of Saeki et al. (3), detailed analyses of GH transgenic pigs revealed that these animals had lower amounts of saturated fatty acids than did nontransgenic controls (14). These differences included a reduction in palmitic (C16:0), stearic (C18:0), and myristic (C14:0) acids, which are associated with hyperlipidemic and hypercholesterolemic diseases in humans. The ratio was 1:1:1 for saturated, monounsaturated, and polyunsaturated fatty acids in these transgenic pigs, which is optimal for the human diet (14, 15). Thus, early in the history of transgenesis, transgenic pork was available containing lowered amounts of saturated lipids. Repeated injections of recombinant porcine somatotropin can also produce altered lipid composition similar to that of the GH transgenic pigs (15). However, neither GH transgenic pigs nor the application of recombinant porcine somatotropin have made their way into commercial pig production, primarily because of the public opposition to this technology.

Ways to overcome the limitations of the microinjection technique based on nuclear transfer now exist. The first cloned piglets were reported as recently as in 2000 (16, 17), and several additional groups have now succeeded in producing cloned piglets. Current cloning efficiency is low but improving (5, 6). With the advent of somatic cloning, it has become feasible not only to add new genes but also to delete unwanted genes (5). For example, pigs have been produced with a knockout for the α-galactosyltransferase gene involved in the hyperacute rejection response to xenotransplantation (18-20). Additional alternatives to microinjection include sperm- or lentiviral-mediated gene transfer. Both procedures seem to give a significantly larger yield of transgenic off-spring than that of microinjection. But reliable data on the long-term expression is missing for both technologies (21, 22).

In contrast to mice and humans, the genomes of the large domestic animal species have not yet been sequenced and annotated completely (5). With the aid of tools developed in the mouse and human genome projects, a first draft of the porcine genome is expected to be available within the next 15-18 months. Nevertheless, genomic sequencing has already led to identification of factors associated with pork quality. For example, the ryanodine receptor gene was identified on human chromosome 19, and a mutation thereof was associated with the malignant hyperthermia syndrome. The homologue was mapped to porcine chromosome 6, where a mutation causes stress susceptibility and impaired meat quality (23, 24). This work led to a DNA test for the mutation that significantly reduced the number of animals suffering from this condition and improved the quality of pork delivered to the consumer. More recently, a dominant mutation has been identified in the PRKAG3 gene, which causes glycogen accumulation in skeletal muscles in Hampshire pigs. This mutation improves the taste of the pork but apparently makes automated processing more difficult (25). Two quantitative trait loci have been identified that have significant effects on muscle mass and fat deposition (26, 27). As sequencing of the porcine genome is completed, pig breeders will gain access to additional genomic loci critically involved in the various aspects of pork quality. The article by Saeki et al. (3) is a first glimpse at what will be technologically possible in the next decade.

The production and characterization of a new transgenic pig line is only the first step in introducing the transgene into a production population. This requires integration into nucleus breeding herds. The nucleus herds must then undergo selection to optimize performance for the desired production trait because the transgene will not give the desired phenotype in every genetic background. A strategy that has been suggested for introgressing a transgene into a nucleus swine herd is backcrossing for three generations before initiating selection of a herd and characterizing the phenotype (28). This approach will enable breeders to identify the most appropriate animal with a sufficient economic merit to compensate for generation costs.

An important aspect in the commercialization of products from transgenic animals is public acceptance. Although the use of transgenic animals for the production of valuable pharmaceutical proteins or functional xenografts is rather widely accepted, genetic modification of animals for human consumption meets significant resistance, in particular, in Europe. The case of Saeki et al. (3) is helpful in this context because it shows a significant improvement in pork quality beneficial for human health. This improvement could facilitate public acceptance of genetically modified food and pave the way for the commercial production of transgenic animals. The lessons learned from the debate on transgenic crops are that “indirect benefits,” such as herbicide or insecticide tolerance, are not enough to win public acceptance of the technology in Europe. With the article by Saeki et al. we have seen an important step toward diversification of agricultural animal products. In light of the improvements in gene-transfer technology, primarily those based on nuclear transfer, and completion of the genome-sequencing projects, it will be possible to deliver better bacon within the next decade or so.