Gene Transfer and Modulation for the Production of Food with Enhanced Quali-Quantitative Values: Potentials, Promises and Achievements

Abstract

Researchers present an overview of the research and achievements in the applications of molecular tools based on gene transfer and gene modulation (gene knock-down and knock-out), aimed at enhancing food production, improving food properties, and producing various valuable compounds for human nutrition. Selected cases of genetically manipulated plants (biofortification and allergen silencing) and animals (fish and livestock) are examined. Promises and accomplishments are considered when giving topic examples of the potentials offered by some applications of molecular biology for obtaining goods, among them are milk with enhanced value, and of their impact on society at large.

Headnote

Summary

We present an overview of the research and achievements of applications of molecular tools based on gene transfer and gene modulation (gene knock-down and knock-out), aimed at enhancing food production, improving food properties and producing various valuable compounds for human nutrition. Selected cases of genetically manipulated plants (biofortification and allergene silencing) and animals (fish and livestock) are examined. Promises and accomplishments are considered when giving topic examples of the potentials offered by some applications of molecular biology for obtaining goods, among them milk, with enhanced value, and of their impact on society at large.

Key words: biofortification, functional foods, gene silencing, gene suppression, health hazards, milk production, human nutrition, transgenic plants and animals, transpharmers

Introduction

This review focuses on the research, promises and achievements of applications of molecular tools based on gene transfer and modulation of genes (knock-down and knock-out) carried out in plants and animals, aim- ing at improving food properties and producing various valuable compounds for human nutrition. The field is huge since a great amount of research has already been developed, and a number of promising results have been achieved. Various documents concerning the state of the art, in particular the regulatory aspects of experimental and market releases of transgenic products, are available on the websites of the institutional organizations involved in agro-food policies, among them the International Ser- vice for the Acquisition of Agri-Biotech Applications (ISAAA) (1), the Food and Agriculture Organization of the United Nations (FAO) (2), the International Centre for Genetic Engineering and Biotechnology (ICGEB) (3), the Joint Research Centre of the European Commission (JRC) (4) and the European Food Safety Authority (EFSA) (5).

This topic is a remarkable example of the controver- sial interactions of science, society and politics which characterize various innovations in biology research. Such innovations, also known as 'bio-objects', leave the labo- ratories and break into our every-day lives bearing their load of promises and concerns. The products of gene transfer and modulation, in fact, are constructed and manipulated biologies potentially useful for enhancing human life quality, balancing on the fine line between the natural and the non-natural/artificial, and challeng- ing conventional natural, cultural, scientific and institu- tional orderings (6). Details of their impact on society can be found in specific literature (7-9) and in our previ- ous works concerning the analysis of consumer accept- ability and perceived risk related to genetically modified organisms (GMOs) (10), and some bio-social implica- tions of biology innovation and food (11,12). Moreover, additional viewpoints can be found on the websites of various associations and non-profit organizations involved in bio-social issues and environmental protection.

Here we report a few selected cases of genetic mani- pulation of plants and animals which we consider most representative and relevant in scientific achievement for their impact on society at large and from a historical point of view. The aim is to build up a portrait of the potentials offered by molecular biology for obtaining food with enhanced value. This portrait is not meant to be a complete magnum opus; conversely, it is intended to stim- ulate readers' interest to further explore this fascinating field. Besides the interest in the scientific aspects, in fact, the production of fortified food, where the properties of food and medicine are strictly linked, points our atten- tion to the deep connection between food consumption, culture and lifestyle, a relationship known since ancient times.

Transgenic Crops for Improved Food Production

The first transgenic plant achievement dates back 30 years, when generations of antibiotic-resistant tobacco (13-14) and petunia (15) were announced at the Miami Winter Symposia of 1983 by three independent groups, followed a few months later by a publication on trans- genic sunflower expressing the bean phaseolin gene (16). Since then, technical improvements and continuous suc- cessful gene transfer have been reported in innumerable plants and, according to the 2011 report of the ISAAA on the Global Status of Commercialized Biotech/GM Crops (1), nowadays 160 million hectares are cultivated with biotech crops, with a globally upward trend. Soya bean, maize, cotton and canola continued to be the principal biotech crops in 2011, and herbicide tolerance has con- sistently been the dominant trait.

Notwithstanding this commercial background, focus- ing mainly on the industrial and feed crops which have been genetically modified for traits useful to farmers as well as biotech companies, there is a constant interest in the scientific world in exploiting gene transfer techniques for improving food properties and producing various valuable compounds for human nutrition. This is reflect- ed by the shift from the first generation of transgenic plants to the second generation (plants with improved quality and reduction of allergenic components) and fur- ther (plant molecular farming, production of renewable resources). Accordingly, many genetically modified plants have been generated with the aim of producing pharma- ceutical and nutraceutical compounds for use by humans, a technology known as biopharming. Vaccine produc- tion in transgenic plants is one of the best known exam- ples of these new generation biotech crops. In particular edible vaccines, produced in edible parts of the plants, in their dual function of food and medicine, have been designed to be easily delivered, and to overcome the drawbacks of classical vaccine manufacturing based on microorganisms and mammalian cells (17).

In some applications of gene transfer for relevant compound production, both plants and animals have been used to reach the same goal, and further experi- mentation would help in identifying the best system, considering efficiency, cost and acceptability of the prod- ucts. This is the case of lactoferrin, a multifunctional protein involved in several biological functions, such as regulation of iron transport, antimicrobial defence and antitumour mechanisms. Of the transgenic systems uti- lized to produce human lactoferrin, besides yeast, trans- genic cows and rice have also been successfully used. The recombinant human lactoferrin (rhLF) is produced on an industrial scale from Aspergillus awamori by Agen- nix (Houston, TX, USA), from rice by Ventria Bioscience (Sacramento, CA, USA) and from transgenic cows by Pharming (Leiden, The Netherlands), and other compa- nies in China and Argentina, as described below. In plants, expression of rhLF has been also achieved in to- bacco, potatoes, tomato, maize, barley and rice (18).

A number of reviews of the scientific literature pro- duced over the years concerning genetically modified plants, including the above-mentioned new generation ones, have been produced; as the total amount of avail- able papers and data have highly increased, they have focused on various different aspects (among the latest papers see 19-24).

In the present review, we concentrate on some se- lected examples concerning gene transfer for improved food production, and in particular for increasing nutri- tional value, an approach also known as biofortification. Biofortification of plant-derived foods appears to be a promising strategy for the alleviation of nutritional defi- ciencies. Various examples of genetically engineered crops in order to increase their nutritional value through im- provement of the level of vitamins/antioxidant com- pounds are already available.

Modification of fatty acid content in food is a rele- vant objective pursued both in animals (as described in the section below) and plants, since scientific evidence has implicated the quantity and/or quality of dietary fats in the development of several illnesses, including cardiovascular diseases, some cancers and arthritis. Ac- cordingly, in canola (Brassica napus) seeds, high levels of ?-3 fatty acids have been accumulated by transferring the genes of the enzymes involved in the fatty acid bio- synthetic pathway (D6 and D12 fatty acid desaturases from the commercially grown fungus Mortierella alpina and the D15 fatty acid desaturase from canola) (25).

Several attempts have been made to engineer higher lycopene levels in tomato fruits. With this aim, the bac- terial crtl gene was introduced in tomato (26) resulting in an unexpected threefold increase in ß-carotene but not in lycopene. Overexpression of lycopene ß-cyclase 03-Ley) was obtained in tomato by introducing via Agrobacterium a construct containing the Arabidopsis ß-Lcy cDNA fused with the tomato Pds promoter, which is up-regulated in ripening fruits. Three transformants showed a signifi- cant increase in fruit ß-carotene content and an orange/ orange-red colour of fruits (27).

Diretto et al. (28) aimed at improving potato with a mini-pathway of bacterial origin, driving the synthesis of ß-carotene (provitamin A) through three exogenous genes: phytoene synthase (CrtB), phytoene desaturase (CrtP) and lycopene ß-cyclase (CrtY). Expression of all three genes under tuber-specific promoter control re- sulted in tubers with a deep yellow phenotype. In these tubers carotenoids increased approx. 20-fold, to 114 ^ig per g of dry mass and ?-carotene 3600-fold, to 47 μ g per g of dry mass, sufficient to provide 50 % of the recommended daily allowance of vitamin A with 250 g (fresh mass) of the transgenic potatoes.

A research project named BioCassava Plus (29) was developed by a public network of researchers, aiming to reduce micronutrient malnutrition by increasing the nu- tritional value of cassava, a staple crop in Africa. The objective of the project was to produce and field test a transgenic variety of TME7 cassava enhanced with ß-caro- tene in Nigeria, such that it contains 40 ^ig of carotene per g of dry mass and 40 μ g of iron per g of dry mass.

Overcoming vitamin deficiency: the case of Golden Rice

Among the crops already produced in the various biofortification projects, Golden Rice has a ringside seat since it is still the paradigmatic example of the potential- ities that a transgenic crop can have, but also of the problems it can cause. This project represents a scientific breakthrough, being the first case of biochemical path- way engineering. Moreover, Golden Rice was intended as a product aiming at solving a major humanitarian world problem rather than to benefit producers (30,31). Nonetheless, it encountered fierce opposition, and there- after few new transgenic Golden Rice lines have been produced throughout the years.

The first paper concerning Golden Rice was pub- lished in Science in 2000 (32). The authors stated that at that moment as many as 124 million children worldwide were deficient in vitamin A. This deficiency causes symp- toms ranging from night blindness to xerophthalmia and keratomalacia, leading to total blindness. Even subclin- ical vitamin A deficiency can have broader consequences in developing countries in terms of child morbidity and mortality. Oral delivery of vitamin A was problematic be- cause of the lack of infrastructure, therefore the supple- mentation of a major staple food, rice, with provitamin A seemed an effective strategy.

In an attempt to develop vitamin-A-producing rice, a project started in 1992, led by Ingo Potrykus of the In- stitute of Plant Sciences at the Swiss Federal Institute of Technology (?ΤΗ, Zürich, Switzerland), together with Peter Beyer of the University of Freiburg, Germany, with the support of the Rockefeller Foundation (New York, NY, USA) and others. Agrobacterium-medmted gene trans- fer was used to introduce the entire ?-carotene biosynth- etic pathway into rice endosperm (the edible part of rice) with different vectors. The vector pB19hpc combined the sequences for a plant phytoene synthase (psy) originat- ing from daffodil (Narcissus pseudonarcissus) with the se- quence coding for a bacterial phytoene desaturase (crtP) originating from Erwinia uredovora, placed under the con- trol of the endosperm-specific glutelin (Gtl) and the con- stitutive CaMV 35S promoter, respectively. The psy cDNA contained a 59-bp sequence coding for a functional tran- sit peptide, and the crtl gene contained the transit pep- tide sequence of the pea Rubisco small subunit: this plasmid directed the formation of lycopene in the endo- sperm plastids. Provitamin A was produced in the ab- sence of heterologous ß-cyclase (which converts lyco- pene to ß-carotene): the absence of lycopene in Golden Rice showed that the carotenoid pathway proceeded be- yond the transgenic end point and thus that the endoge- nous pathway must also be acting (33,34).

The scientists involved in the project intended to make the technology freely available to developing countries but had to face a number of legal constraints coming from pending licences, since part of their financial sup- port derived from the European Community Carotene Plus project, whose industrial partners had rights to pro- tect. An agreement was reached with Zeneca (Fernhurst, UK), corporate sponsors of the Carotene Plus program, and subsequently with most of the Intellectual and Tech- nical Property Rights owners (some 32 companies and institutions hold 70 patents for various technologies used to create the enriched rice), so that it was possible to grant freedom to operate to public research institutions in developing countries to proceed in introducing the trait into local varieties. The cut-off line between human- itarian and commercial use, i.e. the maximum annual in- come for a small-scale farmer to be exempted from roy- alty payment, was set at US$ 10 000 (35).

A substantial improvement in the Golden Rice was achieved by Paine et al. (36), from Syngenta biotechnol- ogy company, who hypothesized that the daffodil psy gene was the limiting step in ß-carotene accumulation. They identified a psy gene from maize and introduced it in combination with the Erwinia uredovora crtl gene, used to generate the original Golden Rice. This Golden Rice 2 (GR2) showed an increase in total carotenoids of up to 23-fold (maximum 37 ng/g) compared to the original Golden Rice, now called GR1 (whose highest provitamin- -A-producing line contained 1.6 ^ig per g of carotenoids in the endosperm), and a preferential accumulation of ß-carotene. ß-Carotene derived from GR2 was effectively converted to vitamin A in humans (37).

An in-depth analysis of the potential economic ef- fects of Golden Rice and the prospective impacts of countries adopting this technology was given by Nielsen and Anderson in 2003 (3S). To have the greatest impact at a low cost, Golden Rice varieties should be adapted for widespread cultivation in Asia and should deliver as much ß-carotene as possible (39); the findings of Stein et al. (40) suggested that related investments were worth- while. The characteristics of Golden Rice may be trans- ferred to locally adapted varieties and ecotypes in rice- -growing countries to limit the reduction of biodiversity. Both the original GR1 and GR2 originated from Japónica rice cultivare; researchers were then backcrossing GR1 and GR2 lines with the Indica varieties popular among Asian farmers. The initial perspective of making Golden Rice cultivare available to farmers in a few years failed, however, and the first field trial of Golden Rice in Asia was only started in 2008 (41).

The most productive rice variety in Bangladesh, BRRI Dhan-29, engineered at the International Rice Re- search Institute (IRRI) in Los Baños, Philippines with the ß-carotene genes from corn, was successfully field-tested at the IRRI in February 2011. Besides BRRI Dhan-29, the IRRI variety IR-64 and the Filipino variety RC-28 were genetically engineered to have greater expressions of the corn gene responsible for producing ß-carotene. Accord- ing to IRRI, if Golden Rice is shown to be safe and to improve the vitamin A status, a delivery program will be designed to ensure that Golden Rice is acceptable and accessible in vitamin-A-deficient communities (42).

A considerable amount of literature and documents concerning the opposition to the Golden Rice project has been published, mostly signed by various institutions and international non-governmental organizations (NGOs), and can be found on the Internet. Besides risks related to the environmental and biodiversity protections, bio- -social concerns and disapproval of the policies adopted for the empowerment of developing countries are the most relevant issues pointed out against the adoption of this crop. One of the most embraced criticisms of Gold- en Rice is that the amount of transgenic rice to be con- sumed during an everyday human diet to meet the daily requirement of vitamin A would be largely unfeasible. Figures reported by scientific papers, however, support results quite divergent from this assumption (36,37), proving the fact that the Golden Rice project has raised a wealth of controversial opinions since its beginning and the debate on the issue is far from being closed.

Still, several years after its first production, whilst the scientific community values Golden Rice as a flag of the humanitarian engagement of public research, oppo- nents, on the contrary, present this bio-object as an ex- ample of technology aimed at further enriching the mar- kets of the already rich countries rather than elevating the economies of the developing countries (43,44). This assertion is well exemplified in the words of philoso- pher Vandana Shiva, the environmental activist and eco- feminist, one of the leaders and board members of the International Forum on Globalization and a figure of the global solidarity movement known as the alter-globali- zation movement: 'While the complicated technology transfer package of Golden Rice will not solve vitamin A problems in India, it is a very effective strategy for corporate take-over of rice production, using the public sector as a Trojan horse' (45).

The promise of resveratrol for healthier transgenic food production

Stilbenes are natural, biologically active phenolic com- pounds occurring in a number of plant families, includ- ing Vitaceae and (within this family) Vitis vinifera L., which is one of the most economically important horticultural crops. In particular, resveratrol (3,5,4'-trihydroxy-ira«s- -stilbene) and viniferins are present in grapevine as con- stitutive compounds of the woody organs (roots, canes, stems) and as induced substances (in leaves and fruit) acting as phytoalexins in the mechanisms of grape re- sponse to pathogens and abiotic stresses (46,47). Several other fruits and vegetables naturally produce stilbenes, as resveratrol has been found in cranberries, blueberries, mulberries, peanuts and jackfruit (48).

Stilbenes also exhibit a broad spectrum of antibiotic and pharmacological activities, and have generated con- siderable interest as nutraceuticals, owing to their di- verse health-promoting properties and their supposed role against cardiac ailments and cancer. The first evi- dence of stilbene synthesis in Vitaceae species was re- ported by Langcake and Pryce in 1976 (49). Since then, intense research aimed at elucidating the role of stilbenes has been carried out, although the topic still needs fur- ther studies. Resveratrol, one of the best known plant secondary metabolites, has also been detected in red wine (it is synthesized in the berry skin and the macera- tion period during winemaking allows for its extraction into the resulting wine) (46).

At the beginning of this millennium, much empha- sis was given to the so-called French paradox by the me- dia, i.e. the fact that French people suffer a relatively low incidence of coronary heart disease, despite having a diet relatively rich in saturated fats: this conflicting data was hypothetically explained by the consumption of red wine, which should decrease the incidence of cardiac diseases (50,51). Thereafter, a great number of studies in model animals and epidemiological observations in hu- mans concentrated on proving the effects of polyphenols and resveratrol in wine, as well as in determining the most suitable dose assumed to produce these beneficial attributes.

Accordingly, moderate wine consumption has been proposed in the prevention of various chronic patholo- gies, in particular those related to cellular oxidative stress, including diabetes, high blood pressure, atherosclerosis, cholesterol reduction and certain cancers (52-55). Other studies (56,57), however, raised doubts about the healthy effects of dietary resveratrol consumption. Recently, works conducted on the beneficial effects of resveratrol at the University of Connecticut Health Center (UCHC, Farm- ington, CT, USA) have been subjected to an extensive re- search misconduct investigation by UCHC itself and the US Office of Research Integrity (ORI, Rockville, MD, USA) (58,59). The significance of this case of fabrication and/or falsification of data, however, seems to be an ex- ample of scientific research fraud rather than an invali- dation of the many studies on the health benefits of re- sveratrol. Possibly, the potentialities of this compound have been overestimated and at the present time, the long-term effects of supplementation of resveratrol and other stilbenes in human diet still need to be ascertained.

Nonetheless, the interesting biological properties and the potential positive effects of these compounds have driven many scientists to study them and to attempt the production of functional food with an enhanced content of resveratrol and related compounds via gene transfer. The role of resveratrol as a phytoalexin has been widely studied, in particular in the defense against fungal patho- gens (60).

Transfer of stilbene-encoding genes to plants unable to produce (or having a limited production of) phyto- alexins has been attempted, starting from the early 1990s. The first gene transfer experiment was performed by trans- ferring a stilbene synthase gene from peanut (Arachis hypogea) to tobacco cells, resulting in de novo resveratrol synthesis after induction with UV-light and an elicitor (61). Genes involved in the biochemical pathway of stil- benes in grapevine were also used in gene transfer expe- riments, such as two grapevine genes coding for stilbene synthase to tobacco. The obtained transgenic tobacco plants were more resistant to Botrytis cinerea infection than the control (62). Since those pioneering works, stilbene synthase genes from grapevine or peanut have been trans- ferred to a number of plants to increase their tolerance/ resistance to plant pathogens or to exploit their positive effects on human health; in some cases both aims were pursued.

Among the gene transfer experiments with the final goal of improving the health protection properties of dif- ferent plant species, genetically modified alfalfa (Medi- cago sativa) was used as a model in applying biotechno- logical approaches to cancer prevention. This transgenic alfalfa was generated with a peanut cDNA encoding re- sveratrol synthase, which produced írans-resveratrol-3-Ο- -ß-D-glucopyranoside (RG), whose constitutive accumula- tion increased resistance to Phoma medicaginis (63). Trans- genic alfalfa or synthetic RG included in mice diets were unable to inhibit the formation of preneoplastic lesions (that directly correlate with the risk of colon cancer and tumour size in humans) in the colon of mice, probably due to the metabolism and biological uptake of the com- pound in the upper gastrointestinal tract, but when these diets were supplemented with the exogenous enzymes ß-glucosidase (64) or a-galactosidase (65), the develop- ment of the preneoplastic lesions was significantly reduced.

Resveratrol was extracted and purified from trans- genic tobacco plants. Having observed an increased ac- cumulation of human breast adenocarcinoma cells in G0 and G] phases of cell cycle in the cells treated with this resveratrol as compared to the untreated cells, it has been concluded that resveratrol from transgenic plants merits further investigation as a potential cancer chemopreven- tive agent in humans (66).

Stilbene synthase genes from Vitis vinifera were trans- ferred to Lactuca sativa, resulting in a transgenic red let- tuce capable of producing resveratrol in amounts higher than red wine (67). Fruit tree species were also geneti- cally modified with genes coding for resveratrol synthe- sis. cDNA encoding stilbene synthase from V. vinifera, transcriptionally regulated by an enhanced cauliflower mosaic virus (CaMV) 35S promoter, was transferred to Spadona pear (Pyrus communis) resulting in piceid and resveratroloside accumulation in transgenic plants (68). A similar approach was adopted in Elstar and Holstein- er Cox apples (Malus domestica), where stilbene synthase gene (Vstl) transfer caused the accumulation of a resve- ratrol derivative, irans-resveratrol-3-O-ß-D-glycopyrano- side (piceid), in fruit tissues (69). The possible influence of the novel biosynthetic pathway on the accumulation of other phenolic compounds naturally present in apple fruit was investigated, and in none of the transgenic apple lines that accumulated piceid was a negative correlation between levels of piceid and the amounts of flavanols, flavonols, phloretin derivatives and hydroxycinnamic acids observed, except for the flavonol content, which slightly decreased (70).

Stilbene synthase production induced by transferred exogenous genes has been obtained in a wide range of plant species, including tomato (71), barley and wheat (72-74), rice (75), poplar (76), pea (77), hop (78), and purple sweet potato (79). These experiments were usually aimed at the production of transgenic plants with increased re- sistance to fungal pathogens, but the positive effects of resveratrol on human health were also considered. How- ever, not all the resulting transgenic lines showed an in- creased tolerance to pathogens when specific bioassays were performed.

While stilbenes can be recovered as an extract from a selected number of plants, these products are not suit- able for many applications in the food/pharmaceutical sectors due to high levels of impurities as well as the overall low concentration of resveratrol and its deriva- tives in the extract (80). Therefore, it is important to de- velop an effective method of producing this compound commercially. To deliver a highly defined and enriched resveratrol product, hairy root cultures of peanut were studied as a bioproduction system for resveratrol and associated derivatives. In this system, 99 % of the total resveratrol produced was secreted into the culture me- dium at levels of 98 ^ig per mg of the medium dried extract (80).

Given the increased interest in these compounds and the increasing number of experiments aimed at the gen- eration of transgenic plants overexpressing them, some comprehensive reviews have recently been published, focusing on the role and activity of resveratrol (47), its molecular engineering in plants (81,82), and methods for obtaining it, from plant extraction to chemical synthesis and biotechnological production (83,84). In these papers gene and promoter options are discussed, as well as fac- tors modifying transgene expression and epigenetic modi- fications, the incidence of these compounds in plant me- tabolism and development, and the use of biotechnology through recombinant microorganisms and plant cell sus- pensions.

Transgenic Animals for Improved Food Production

Gene transfer in animals was pioneered at the end of the 1970s, starting with a mouse expressing foreign DNA sequences of tumour virus SV40 (85), and nowa- days this technique has been successfully applied in a considerable number of farm animals such as rabbit, pig, sheep (86), cattle (87) and goat (88).

Direct microinjection of genetic material into the pro- nuclei of fertilized one-cell eggs to be implanted into the oviducts of pseudopregnant surrogate female mice has been the first effective strategy developed (89) and, even though laborious, costly and hampered by relatively low efficiency and species-dependent embryo survival, is still reported as the most accepted method of production of transgenic animals (90). Alternatively, starting from the pioneering studies developed in the 1970s based on retro- virus vectors (91), lentiviral vectors have been efficiently used for microinjecting fertilized eggs, unfertilized oocytes and zygotes, and nowadays lentivirus-mediated trans- genesis is the most strongly emergent technology for de- livering genes as well as for transfecting synthetic small interfering RNA (siRNA) molecules aimed at targeting gene knockdown (90,92). The combination of somatic cell nuclear transfer, an established tool to yield copies of se- lected individuals in different animal species, with ge- netic engineering is currently reported as the most suit- able method for generating transgenic large animals for both agricultural and biomedical applications (93).

It is worth remembering that male germinal cells (spermatozoa and spermatogonial stem cells) have been used to direct the transfer of exogenous DNA into fe- male cells for generating transgenic animals (94). Embry- onic stem cell-mediated gene transfer has also been de- veloped (95) by inserting a desired DNA sequence via homologous recombination into an in vitro culture of these totipotent cells, which are then incorporated into an em- bryo at the blastocyst stage of development. DNA con- structs can also be transfected by electroporation into fe- tal cells, such as fibroblasts, which can thereafter be used to clone transgenic animals by nuclear transfer (96). Com- bination of classical breeding with gene transfer may also be used, as in transgenic cows which, besides insertion of a desired gene into embryos, can be produced by mat- ing wild type cows with genetically modified bulls for the generation of transgenic progeny (97).

To date, according to the results obtained in animals relevant to food production, gene transfer seems to be a costly strategy, and not as efficient as initially promised, compared with conventional selection. Moreover, trans- genic farm animal production for biomedical applications encountered more acceptability and provoked less pub- lic concern, thus influencing the industrial application of GM animals. Consequently, gene transfer has primarily been used for biomedical rather than agricultural appli- cations. In medicine, genetically modified pigs have been adopted as organ sources for xenotransplantation (98). The generation of transgenic mice capable of mimicking human genetic diseases provides a suitable whole ani- mal model for studying human genetic diseases in order to develop pathological and pharmacological studies (99). The production of drugs for pharmaceutical use is an- other relevant application of transgenic animals, in par- ticular to overcome the limitation of recombinant bacte- rial systems, which are less suitable for producing complex proteins such as monoclonal antibodies or coagulation blood factors. Moreover, compared to plants, the high yield and low-cost production as well as the high qual- ity of the proteins obtained are the features which make animal systems appealing tools (100). The most common system is milk from transgenic farm mammals; never- theless, chicken egg white and blood, seminal plasma, urine, silk glands and insect larval haemolymph have also been exploited (100).

As for food improvement, animals have been genet- ically modified to obtain improved livestock and quali- -quantitatively enhanced food sources. Productivity traits, such as feed conversion, rate of gain, reduction of fat, and improved quality of meat are the most pursued ob- jectives (101). The productivity traits are however quan- titative, thus controlled by numerous genes, of which only a few are presently known. For this reason, besides the current low level of public acceptance, the progress of agricultural applications of livestock transgenic technol- ogy is less developed compared to biomedical applica- tions. Among the hundreds of quantitative trait loci (QTL) already mapped in livestock, two mutations underpin- ning the QTL have been identified in dairy cattle: the first concerns the 1DGAT1 (acyl-CoA:diacylglycerol acyl- transferase) locus on chromosome 14 as a gene contrib- uting to fat composition in milk (102) and the second concerns the ABCG2 (ATP-binding cassette, subfamily G (WHITE), member 2) locus on chromosome 6 as a gene contributing to fat and protein concentration in milk (103).

More advanced is the knowledge of monogenic traits of economic and biological interest, and a number of caus- ative mutations have already been identified, as reported in the valuable database Online Mendelian Inheritance in Animals (OMIA) (104), where numerous single-locus traits are described in 192 animal species other than hu- man and mouse, including food sources as interesting as cattle, pig, sheep, goat, chicken and horse, according to the different food ethics of various countries, which ei- ther assign the status of food source to animals or desig- nate their consumption as a taboo (105).

In the near future, the achievements of genome proj- ects will offer useful information for better understand- ing of the most economically important genes in live- stock species in order to accomplish the selection for desirable traits, or conversely, against undesirable traits (106), and in the main species, like bovine ones, genome sequencing has already been accomplished (107).

Growth enhancement in transgenic animals

Growth enhancement is one of the most pursued applications of transgenic techniques. Pigs, fish and cat- tle have been engineered in an attempt to provide more food per animal, generally by transferring growth hor- mone genes (108). Growth hormone (GH) is a peptide hormone naturally synthesized, stored, and secreted by the pituitary gland, which stimulates growth, cell multi- plication and regeneration (known as anabolic effects) in humans and other animals. Over- and under-production of GH are related to relevant pathologies.

Production of superfish has received the most atten- tion because of its success in generating marketable prod- ucts in shorter periods of time and with lower produc- tion costs, and has been applied to a wide range of fish species including the coho (Oncorhynchus kisutch), Atlan- tic (Salmo salar) and Japanese (Oncorhynchus masou) sal- mons. As detailed in Maclean and Laight's review (109), the growth enhancement of fish species can be dramati- cally improved by transgenesis based on the GH gene: in the Atlantic salmon and coho salmon, more than ten- fold larger G0 fish than control counterparts were ob- tained, and in Tilapia sp. up to more than threefold mass increase was recorded. Results in carp, northern pike and channel catfish were not so dramatic, but still rele- vant, possibly because of the considerable strain selec- tion over many hundreds of years of human exploitation rather than because of the failure of transgenesis.

Some North American multinational companies have already developed transgenic salmon, trout, and tilapia designed to grow faster than their conventional siblings. This is the case of the AquAdvantage® Salmon produced by AquaBounty Technologies (Waltham, MA, USA), which presents the product with the following words: 'AquAd- vantage® Salmon (AAS) includes a gene from the Chi- nook salmon, which provides the fish with the potential to grow to market size in half the time of conventional salmon and, in all other respects, AAS is identical to other Atlantic salmon' (110). This salmon, which repre- sents a profitable solution to increasing fish demand in the coming years, will be recorded as the first transgenic animal for human consumption approved by Food and Drug Administration (FDA) (111). A lengthy authoriza- tion process started in 1995, when AquaBounty Technol- ogies applied for official US Government approval to develop this salmon commercially. In 2010, the FDA an- nounced that there was enough information available to review the GM salmon, and in May 2012 it completed its environmental assessment. In December 2012, a draft was published declaring that the production of the GM fish is unlikely to have any detrimental impact on the wider environment, opening the doors for commercialization and yet again raising a strong anti-GMO reaction (112).

Initially, GH genes from mammals, principally hu- man, bovine and rat, were the first exogenes used for developing the superfish under the control of the metal- lothionein ? promoter. Recently, sequences derived from fish itself ('all fish' genetic material) carrying both pro- moters and genes from fish origin have been employed on the basis of a presumed higher likelihood of accept- ability by consumers, as in the case of cisgenic plants (113). Oppositions to genetic manipulation of animals, as for plants, however, seem to ignore this assumption. In 2001, the European Patent Office (EPO) granted its first patent (Patent No. 0578653) on genetically engineered fish concerning a gene construct for production of trans- genic fish produced by Canadian Seabright Corporation Ltd., Toronto, Canada (114). The construct carried a pro- moter sequence of the antifreeze protein derived from ocean pout. This promoter proved to provide better gene expression and regulation in the fish (115). For instance, transgenic Atlantic salmon, generated with all fish GH chimeric gene constructs carrying this promoter linked to a chinook salmon GH, presented an amazing growth rate since, when one year old, the average increase was 2- to 6-fold and the largest transgenic fish was 13 times bigger than the non-transgenic controls (116).

Transgenic superfish have been studied in depth and specific tests have been used to measure their perfor- mance, including manoeuvrability, speed of escape re- sponse, sprint speed and propulsive efficiency. Some evi- dence to support the view that growth-enhanced trans- genic fish may be inferior to their wild-type counterparts has been published, since in transgenic rainbow trout a behaviour related to an increased prédation mortality has been seen, and in transgenic coho salmon a lowered swimming performance (117,118) has been observed. Ap- plication of gene transfer to fish is relatively simple, but the 'mobility' of transgenic fish and the consequent need for containment are related problems and, besides strict containment measures, sterilization has been proposed to overcome this disadvantage (109,119).

GH gene transfer has also been exploited in pigs and resulted in transgenic animals having faster mass gain, higher (17 %) efficiency in converting feed into meat and reduced (down to one-fifth) fat in the carcass compared to the wild-type counterparts. However, various delete- rious effects have also been observed (120). This is the case of a pig named Beltsville pig after the Agricultural Research Station in Beltsville, MD, USA, where it was generated, and nicknamed super-pig for its large size. This animal was produced by zygote microinjection with a construct carrying an ovine metallothionein la-ovine growth hormone fusion gene. High-level expression of the transgene resulted in a dramatic reduction in carcass fat with a concomitant increase in carcass protein and moisture content as compared to the control littermates. However, the high expression of this transgene due to its ineffective regulation hindered these results, and vari- ous health problems (lethargy, lameness, uncoordination, exopthalmus, gastric ulcers, severe synovitis, degenera- tive joint disease, pericarditis and endocarditis, cardiomeg- aly, paraketosis, nephritis, and pneumonia) related to its enormous size were the side effects in this experimental pig, which at the end could not support its own mass (120, 121). Such severe problems were thought to be the result of the constant high level of circulating GH, but at- tempts to control expression by the use of inducible pro- moters have not yet been successful. Consequently, the super-pig could never be used for ethical reasons, was therefore put down to end its suffering and served as an example for critics of transgenic animal research (122).

In sheep, transgenic GH lambs did not present fast- er growth nor more efficient feed conversion compared to controls; however, they were much leaner and pre- sented a lack of body fat, this latter trait being attributed to the result of hyperglycemia and glycosuria (123,124). Transgenic lambs with elevated GH also had a number of other pathologies, including joint problems and de- generative kidney disease (120).

Overexpression of GH genes in transgenic mice has been applied in order to understand the effects of long- -term GH excess. In these models, bovine, ovine or rat GHs (i.e. hormones with actions closely resembling, if not identical to, those of endogenous mouse GH) exhib- ited growth enhancement, increased adult body size, re- duced lifespan and various endocrine and reproductive abnormalities, i.e. a variety of direct and indirect actions at the hypothalamic, pituitary, gonadal, and reproduc- tive tract levels (125).

Quality improvement of food

Production of food with improved quality, as a re- sult of enhanced/enriched/reduced contents of specific components, is another main goal of livestock genetic improvement. Modification of polyunsaturated fatty ac- ids (PUFAs) is one of the most relevant goals, as clinical studies have shown that long-chain ?-3 PUFAs have a beneficial effect on human health, whilst high levels of n-6 PUFAs in human bodies are closely related to cancer, cardiovascular diseases and mental disorders (126). Since mammals lack n-3 desaturases, the n-3 and n-6 PUFAs are not interconvertible. Accordingly, products from sea fish are the main dietary source of n-3 fatty acids for hu- mans. For this reason farmers and the livestock produc- tion industry feed animals with flaxseed, fish meal or other marine products for enriching foods, contributing on the other hand to the depletion of marine fish stock as well as running the risk of potential contamination of fish products with mercury and other chemicals (96).

Following a promising experiment where exogenous expression of a humanized fat-1 gene (hfat-1) encoding an n-3 fatty acid desaturase from Caenorhabditis elegans in mice resulted in a significant increase of n-3 fatty ac- ids in various tissues and in milk, as well as in a sharply decreased ratio of n-6/n-3 fatty acids (127), this exogene was transferred in pig and cow to produce healthier live- stock. In both experiments, fetal fibroblast cells were used for gene transfer and transgenic animals were cloned from these cells. In piglets, in the major tissues (muscle, liver, kidney, heart, spleen, tongue, brain and skin) of the vari- ous founder animals recovered, and in their offspring, n-3 PUFAs were significantly increased, while n-6 PUFAs were decreased, resulting in a greatly reduced n-6/n-3 ratio (96).

In cows, only a transgenic calf was generated; how- ever, this animal exhibited promising features. Analyses of fatty acids from ear tissues, in fact, showed that all major n-3 fatty acid peaks were elevated, whilst all ma- jor n-6 peaks were at a lower level, thus proving suitable expression of n-3 desaturases (128). Moreover, this ani- mal grew healthy and, after artificial insemination, natu- rally delivered a healthy calf. The content of n-3 PUFAs in milk was significantly increased and the ratio between n-6 and n-3 was reduced fourfold compared to the non- -transgenic counterparts. These results showed that the transgenic cow was capable of normal reproduction, the transgene was properly expressed in the mammary gland and the n-3 fatty acids were enriched in the milk (128).

Milk production by transgenic animals

Milk-producing transgenic animals are the objects of extensive research aimed at manufacturing compounds to be available in, or extracted at low cost from, their milk. Transgenic animals containing human proteins in their milk are also known as transpharmers and various animal species have already been adopted as transpharm- ers (129).

The expression vector carrying the gene encoding for the protein of interest fused to milk-specific regulatory elements is generally introduced into the germline of the chosen species via pronuclear microinjection of one-cell embryos or transfection into a primary cell population suitable for somatic cell nuclear transfer. Mice are main- ly used for experimental assays and for testing the con- structs prior to generating larger transgenic founder ani- mals. Transgenic rabbits are not suitable for large-scale production because of their small lactation yield and la- bour-intensive milking, whilst sows have been success- fully used for the considerable amount of milk (100-200 L) they can produce. Transgenic ruminants are obvious candidates since thousands of years of genetic selection have yielded breeds of sheep, goats, and cattle that can produce prodigious quantities of milk (129).

More than 60 therapeutic proteins, including plasma proteins, hormones, monoclonal antibodies, vaccines, cyto- kines, enzymes, fibrinogen and insulin have already been produced experimentally from the milk of transgenic cows, sheep and goats (97). Amongst them is the anticoagulant antithrombin (ATryn), the first biological drug from ge- netically engineered animals which was manufactured by the US company GTC Biotherapeutics (Framingham, MA, USA) from the milk of a transgenic goat and ap- proved in February 2009 by the US Food and Drug Ad- ministration (see the FDA archive, 130). Transgenic milk has also been exploited for industrial applications, like the high-value polymers produced in goat milk as a re- sult of exogenous expression of the silk gene from spider (131).

Gene transfer has been used for improving livestock growth, and for health and survivability of milk-fed new- borns. In pigs, for instance, where milk production is a limiting factor for piglet growth, overexpression of bo- vine ?-lactalbumin, which plays a key role in the lactose synthesis and regulation of milk volume, resulted in in- creased milk lactose (thus an enhanced carbohydrate source) associated with a 20-50 % increase in milk yield. As a result, the growth and survival of suckling piglets improved significantly (132).

The introduction of new antimicrobial properties into milk is a desirable aim to provide passive immunity for the suckling young, but also for humans consuming the milk. This was the goal of a work aimed at overexpress- ing the human form of the antimicrobial protein lyso- zyme in the goat's milk, which was used for feeding pig- lets as human models and resulted in a beneficial effect on their intestinal microflora (133).

The mammary gland of dairy cattle is considered ideal for large-scale production of heterologous proteins due to its large capacity for protein synthesis, efficient secre- tion, and low feed and housing costs of dairy animals compared to in vitro fermentation or tissue culture sys- tems (18). Accordingly, a number of research projects have concentrated on transgenic milk production both for medical and nutritional properties. As functional food, engineered milk links together the attributes of medici- nal and nutritional, becoming a medicinal food. Bovine milk, in fact, is an important food for humans, with a consumption of milk and milk products per capita vari- able among world regions.

Cow's milk is a source of lipids, proteins, amino ac- ids, vitamins and minerals and contains immunoglobu- lins, hormones, growth factors, cytokines, nucleotides, peptides, polyamines, enzymes and other bioactive pep- tides (134). In western societies, however, a decreasing trend in milk consumption has been noted during the last decades, probably due to claimed negative health ef- fects that have been attributed to milk and milk prod- ucts because of its high saturated fatty acid content (134). Thus improved milk, particularly as far as qualitative at- tributes are concerned, is an attractive goal. Here we con- sider an improvement based on additional gene transfer, whilst some interesting perspectives of gene modulation and silencing are reported in the next section.

Reducing the susceptibility of livestock to pests and diseases via genetic engineering has long been an ambi- tious goal, which would also result in improved produc- tion and reproductive performance and could ultimately provide safer food products of superior quality (135). This concerns, for example, mastitis, a bacterial infection of the mammary gland, which is one of the most costly diseases in cattle breeding, and can also result in death. In dairy cattle, mastitis is mostly caused by Staphylococ- cus aureus infection, a pathogen particularly hard to con- trol with antibiotic treatments. A transgenic strategy has proved to be promising for the prevention of mastitis in cattle by expressing exogenous lysostaphin, an endopep- tidase with lytic properties on a Staphylococcus cell wall. In fact, transgenic cows producing lysostaphin in their milk showed a high degree of protection when challenged with S. aureus. Moreover, increased antimicrobial proper- ties in the milk of mice associated with the overexpres- sion of the endopeptidase lysozyme or lactoferrin pro- vided evidence for the potentials offered by this approach to prevention of mastitis in dairy cattle (135).

As for the enhancement of the functional properties of dairy milk via a transgenic approach, some promising results regard the production of milk with less lactose or cholesterol (136). New Zealand has been a leader in re- search concerning transgenic milk production and, since 2000, scientists at AgResearch (137) have been success- fully producing transgenic cows that yield modified milk containing therapeutic proteins to treat human diseases. Various experimental field trials, in proper containments, have been approved by the Environmental Risk Manage- ment Authority of New Zealand (ERMA). Among the various results, qualitatively improved dairy milk com- position with higher casein content (up to 20 % more) was achieved by transferring additional copies of the genes encoding bovine ?- and κ-casein (138). This result was quite interesting, considering the relevance of casein in milk composition, representing 80 % of the overall proteins, having important nutritional and processing properties and therefore being one of the prime targets for milk composition improvement. Moreover, this was the first proof that transgenic technology could be used to modify milk composition in cows following previous research focused on transgenic cows to produce thera- peutic recombinant proteins.

The production of cheap surrogates for human breast milk for the newborn has been a relevant goal in the food industry and as an alternative to infant formula, transgenic technology for producing cow's milk with in- creased similarity to the properties of human milk has been pursued by a few companies in different parts of the world. Besides the absence of the species-specific quali-quantitative composition of minerals, carbohydrates, proteins, fat, prebiotics and antibodies which make hu- man milk unique to a baby's nutrition, hard digestibility and allergenicity are the most relevant drawbacks of cow's milk (139).

Human lactoferrin, the iron-binding protein also pre- sent in human breast milk and in particular in colostrum, has been regarded as a suitable candidate to enrich cow's milk on the basis of its recognized physiological proper- ties, which are anti-inflammatory and have antipatho- genic functions and roles in iron absorption and/or ex- cretion in newborns as well as in the promotion of intestinal cell growth (18,135,140). In milk, recombinant human lac- toferrin (rhLF) has been overexpressed in concentrations high enough to meet the needs of human children, with the goal of obtaining functional dairy milk with increased protection against infections and improved gastrointes- tinal health properties. The obtained recombinant protein was highly similar to the human natural counterpart for its most prominent biological activities, i.e. iron binding and release and antibacterial activity, thus paving the way for safe usage of rhLF in humans (141). Moreover, that work proved the potential of transgenic cattle as biore- actors in terms of capacity (140). The milk was obtained from the female offspring of one of the first notable trans- pharmer animals, Herman the Bull, which was engineer- ed to carry a gene for lactoferrin by Gen Pharm Inter- national (Mountain View, CA, USA) (142). Herman fathered eight calves in 1994, following a breeding program es- tablished at Gen Pharm's European laboratory (Leiden, The Netherlands); in 2004 was euthanized for stopping osteoarthritis sufferance and, after taxidermy mounting, exposed as bio-object icon in the Naturalis Biodiversity Center, Leiden, the Netherlands (143).

In 2011, the media gave resonance to two experiments occurring in laboratories in different parts of the world (Argentina and China), both claiming the first achieve- ment, aiming at producing transgenic cow's milk using human genes that allow the animal to produce the equi- valent of a (human) mother's milk. On April 2, 2011, The Telegraph announced that the Chinese Academy of En- gineering had led a research which generated cows pro- ducing human lactoferrin (144). What was new in this research, according to official statements, was that, after seven years of research, by using a property technology, the produced milk contained the highest level of lacto- ferrin in the world. The Chinese Ministry of Agriculture approved the testing on genetically modified dairy cows for human breast milk, and official media reported that this milk would become the first transgenic animal prod- uct in commercial use with prospects for use in infant formula milk powder, health foods and medicines.

A few days later, the Argentinean National Institute of Agricultural Technology (INTA; Buenos Aires, Argen- tina) announced that a cloned cow, named Rosita ISA was the first cattle born in the world with two human genes coding for proteins (lysozyme and lactoferrin) present in breast milk (145). The lysozyme is an enzyme present in (human) breast milk at high concentrations during the first week of lactation. Lactoferrin exists in all mammals but is specific to each species, and therefore bovine lactoferrin does not affect humans. 'As an adult, the cow will produce milk that is similar to humans', INTA's statement said. The goal was to increase the nu- tritional value of cow's milk with lactoferrin, which pro- vides infants with antibacterial and antiviral protection and improves iron absorption, and lysozyme, which is also an antibacterial agent.

Transgenic milk production, and in particular the above-reported case of AgResearch in New Zealand, has been rightly considered a remarkable case of the impact of scientific innovation on society, and has been a case study in analyzing the contested representation of the re- search in its promotion, its governance, and the opposi- tion sparked amongst environmental/antigenetic modifi- cation groups, including the Maori community (146). Quite interesting, and worth devoting significance to, was the opposition against this research, and the government en- vironmental risk management authority (ERMA) ap- proval of the experimental field trials, conducted by the New Zealand women's organization Mothers Against Ge- netic Engineering in Food and the Environment (MADGE), a 'network of politically non-aligned women who are ac- tively resisting the use of genetically engineered mater- ial in our food and on our land', led by artist Alannah Currie (147). In particular, two campaigns, a masquerade in the parliament building and billboards displayed in Wellington and Auckland, New Zealand, based on a strong, creative and provocative symbolic communica- tion, were renowned for their astonishing approach and related media reactions. They were meant, really, to con- dense metaphors and visual and emotional excesses in order to provoke a public debate about the social and cultural ethics of genetic engineering in New Zealand. The topic, on the other hand, was quite inspiring, consi- dering emotional and cultural implications concerning ani- mal milk and mother's breast milk.

Gene Modulation and Silencing for Improved Food

Molecular techniques for food improvement have mostly been based on gene transfer into target genomes. Nonetheless, gene silencing of undesired traits would be a promising tool, and it is expected that besides (or maybe instead of) improvement based on traditional gene trans- fer, further research will be focused on adopting this stra- tegy in plants and livestock.

Exogenous RNA-based gene regulation

Besides the various kinds of ribonucleic acids (RNA) present in organisms, such as messenger (mRNA), ribo- somal (rRNA) and transfer (tRNA), many eukaryotes have an additional, highly abundant class of non-coding RNAs (20 to 30 nucleotides) generally referred to as small RNAs (sRNAs) (148). In the cell, sRNAs have various functions, such as regulation of gene expression, development and chromatin structure, and represent an important plant de- fense mechanism against viral infections and transpo- sons. sRNAs are distinguished in two main classes: micro RNAs (miRNAs) and small interfering RNAs (siRNAs). The first derive from single-stranded endogenous RNA transcripts folding themselves up to produce imperfect double-stranded stem loop precursors, whilst siRNAs are processed from long, perfectly double-stranded RNA (dsRNA) precursors from endogenous (irans-acting siRNAs, natural antisense transcript-derived siRNAs, heterochro- matic siRNAs) or exogenous sequences, for example from viruses, which are cleaved in the cell to short RNA frag- ments (149).

Numerous studies have demonstrated that siRNAs in cultured cells of plants and animals can trigger highly efficient gene silencing through degradation of the en- dogenous mRNA, whose sequence is complementary to the siRNAs. The most commonly applied methods for gene silencing based on siRNAs are virus-induced gene silencing (VIGS), RNA interference (RNAi), miRNA as in the case of artificial miRNA (amiRNA) and miRNA-in- duced gene silencing (MIGS) (150).

The process of introducing RNA molecules into cells to suppress the expression of a gene of interest, for ob- taining RNA interference (RNAi), is nowadays a crucial tool in functional genomic studies, and various technol- ogies have been developed. In plants, several constructs adapted to Agrobacterium-medmted gene transfer or vi- rus vectors are available (151), whilst in animals trans- fection via liposomes, electroporation and viral gene transfer via recombinant viral vectors based on retro- virus, adeno-associated virus, adenovirus, and lentivirus are the most applied strategies (152). Further develop- ment in the knowledge of plant and livestock genomes is expected to enlarge the list of candidate genes to be modulated for useful application in enhanced food pro- duction.

RNAi in plant-derived food

In plants, RNAi can be used to improve plant nutri- tional value, as in the case of improved content of essen- tial aminoacids. Lysine, for instance, is an essential amino acid necessary for human health, which has to be intro- duced through the diet since humans are not able to syn- thesize it. In the selected maize lines, Illinois High Pro- tein (IHP), whose extremely high protein value is however affected by no lysine content against a too high level of zein, RNAi anti ?-zeins have been obtained by Agrobac- ienwiw-mediated transfer of a construct carrying inverted repeated gene sequences encoding RNAs for silencing the 22- and 19-kDa a-zeins (153). Analysis of protein content showed a change in protein composition, in particular concerning the ratios of zein and non-zein fractions of total proteins. Moreover, whilst an increase of lysine re- sulted in seeds with enhanced nutritional value, an incom- plete ?-zein reduction maintained a desired property of the kernel, i.e. a vitreous, hard endosperm. This result proved that RNAi strategy is effective for obtaining ker- nels where three relevant traits (high-protein and high- -lysine contents and vitreous, hard endosperm) are com- bined, i.e. a result that has never been achieved by con- ventional breeding.

In food improvement, RNAi can also be applied to silence the genes coding for antinutritional or harmful components. In plants, a very attractive application of gene silencing is the reduction of the allergenic proper- ties of plant-derived food, and some interesting research has already been developed in this framework. In peanut, RNAi technology was used to eliminate the immunodo- minant Ara h 2 protein, the major allergen in peanut by transferring into peanut hypocotyls, via Agrobacterium, the inverted repeats of the coding region of Ara h 2 genomic DNA cloned in the a RNAi-inducing plant transforma- tion vector pHANNIBAL. Crude extracts of several trans- genic seeds of the T0 plants showed the absence of this protein and their allergenicity was checked as IgE-bind- ing capacity in the sera of patients allergic to peanuts. Showing, by ELISA test, a significant decrease in the IgE- -binding capacity of transgenic seeds compared to the wild- -type, the feasibility of alleviating peanut allergy by RNAi technology was proved (154).

Allergenicity cross-reaction between pollen and ed- ible crops is known to afflict the population who suffer from allergies. This is the case of people who are allergic to birch pollen and also become allergic to apple fruit because of the immunological similarities between the major allergen in birch, Bet ν 1, and the major allergen in apple, Mal d 1. To overcome this suffering, inhibition of Mal d 1 expression in apple plants by RNA interfer- ence has been successfully attempted (155). A T-DNA con- struct coding for an intron-spliced hairpin RNA carrying a Mal d 1 -specific inverted repeat sequence, separated by a Mal d 1 -specific intron sequence, was introduced into apple leaf explants via Agrobacterium. This construct effi- ciently silenced the target gene, as confirmed by quanti- tative real-time PCR. Moreover, silencing was unaffected by grafting and remained stable for more than 3 years, throughout all the developmental stages (156). Among other interesting examples of successful application of RNAi for producing crops with reduced/absent allergen- ic properties, it is worth mentioning soya bean, where transgenic suppression was successful in down-regula- tion of the major allergene P34 accumulation in seeds, with no apparent negative consequences on the normal seed development (157), and tomato, where the genes for allergens Lyc e 1.01 and Lyc e 1.02 were successfully silenced (158).

It has been observed that some substances that are toxic for humans, are, however, very important for plants. This is the case of gossypol, a natural phenol derived from the cotton plant (genus Gossypium) with known anti-insect properties. Whilst it has been estimated that global cottonseed production would potentially provide the protein requirement for half a billion people per year, this plant is under-utilized as a food crop because of the presence of toxic gossypol in the whole cotton plant, in- cluding seeds. Accordingly, the elimination of gossypol from cottonseed has been a long-standing goal of breed- ers.

RNAi was successfully used to break down gossypol biosynthesis in cottonseed by targeting a gene coding for the key enzyme ?-cadinene synthase (159). Transgenic cotton was generated by transferring via Agrobacterium a hairpin RNA construct carrying a trigger sequence of the Gossypium hirsutum ?-cadinene synthase gene, under the control of a highly seed-specific cotton ?-globulin ? gene promoter. The level and stability of gossypol in cotton- seed and other tissues were determined in Tj and T2 plants and seeds. In seeds, gossypol quantity was signi- ficantly reduced in a stable and heritable manner. More- over, since the levels of gossypol and related terpenoids in the foliage and floral parts were not decreased, and thus their function in plant defense against insects and diseases remained unaffected, this strategy proved to be interesting for producing plants with both good agro- nomical characteristics and enhanced food properties.

RNAi in animal-derived food

Applications of gene down-regulation or silencing may also open up interesting perspectives in animal-derived food. Myostatin null mice, for instance, generated by gene targeting, showed a dramatic and widespread increase in skeletal muscle mass compared to wild-type count- erparts, due to an increased number of muscle fibres without a corresponding increase in the amount of fat (160). Accordingly, reduction of myostatin expression could be an attractive application of gene silencing for enhancing muscle development in the livestock industry, in particular for beef animals, and would be a promising alternative to the use of GH genes which, as pointed out above, gave disappointing results in mammals.

Myostatin, in fact, also known as growth differentia- tion factor 8 (GDF8), proved to have an inhibitory effect on muscle differentiation and growth, as animals lacking myostatin or treated with substances that block the bind- ing of myostatin to its receptor showed significant muscle hypertrophy (161). This protein has been the object of various studies and myostatin genes have been charac- terized and found to be highly conserved among ver- tebrate species. Two breeds of cattle, Belgian Blue and Piedmontese, which were characterized by increased muscle mass because of their increased ability to convert feed into lean muscle (double-muscling trait) proved to carry mutations (nonsense and frame shift mutations) in the myostatin coding sequence.

It should be pointed out, however, that the selection of double-muscled animals has resulted in a huge increase in the incidence of dystocia, i.e. difficult calf delivery, as in double-muscled Belgian Blue cows, Caesarean section is routinely required (over 90 %) for reducing calf losses (162). The increased muscle mass and feed efficiency, however, have been evoked as appealing features, balanc- ing such a drawback (160).

Nonetheless, in mouse, another interesting selective gene targeting strategy to interfere with the myostatin pathway has been achieved, and it could open the door to further application in cattle production where different expression of traits between the sexes would be desirable. This strategy is based on the generation of transgenic ani- mals for the Y-chromosome-linked muscle-specific expres- sion of a dominant negative myostatin pro-domain, which resulted in the production of males showing a 5-20 % increase in skeletal muscle mass, whilst females were non- -transgenic and thus not affected in their growth pheno- type (135).

Molecular strategies would also be promising tools for preventing some fatal diseases, which also have del- eterious effects in humans through ill animal consump- tion, like the neurodegenerative prion diseases or trans- missible spongiform encephalopathies, including scrapie and the mad cow disease (BSE). In these syndromes, a misfolded isoform of the cellular prion protein (PrP) proved to accumulate and act as a novel infectious agent; thus, the introduction of mutated prion protein genes, gene knockout or RNAi-mediated knockdown of PrP ex- pression would be expected to produce livestock ani- mals resistant to prion diseases. Some research has been done in mouse, and initial studies in sheep, goats and cattle seem promising; in vitro assays with brain tissue homogenates derived from PrP-deficient cattle produced via PrP knockout demonstrated resistance to prion pro- pagation and the cattle were apparently normal in all ana- lysed aspects (135).

As for the improved nutritional value of milk, with its relevant implications for health of the many human adults who lack intestinal lactose-hydrolyzing enzyme activity, an interesting application of RNAi would be the reduced presence of lactose based on ?-lactalbumin ex- pression knock-down. Compared to transgenic knock-out, which has already been obtained in experimental mouse and resulted in the production of lactose-free milk, down- -regulation would produce milk with the adequate lac- tose content to allow osmotic regulation and concentra- tion of milk (163). Similarly, RNAi knock-down expression of the ß-lactoglobulin gene would reduce the allergeni- city of cow's milk (135).

Concluding Remarks

As described in this review, the opportunities offered by molecular tools both in plants and animals for pro- duction of food with enhanced quali-quantitative values are quite numerous, and notable. Moreover, the amount of new information, in particular the data continually ac- cumulated with the genome projects, predicts even greater potential. However, according to the state of the art, the significant amount of research efforts fulfilled in the last decades seems to have produced more promises than concrete achievements to offer to the market.

A series of issues can be mentioned as explanations for this drawback: first of all, the considerable amount of resources and time required in order to acquire reli- able knowledge on gene involvement in biological path- ways related to food properties and to develop sound techniques for applying the knowledge in the produc- tion of goods for human nutrition. Besides, there is a se- ries of central issues, including risk evaluation process, bureaucratic approval, regulatory procedures and public acceptability, which complement, whilst often delaying, scientific achievements, as exemplified by the above-de- scribed cases of Golden Rice, AquAdvantage® Salmon and the surrogates for human breast milk for the newborn.

Acknowledgements

This review was developed in the framework of a cooperation within COST Action IS1001 'Bio-objects and their boundaries: governing matters at the intersection of society, politics, and science' (http://www.cost.eu/doinains_ actions/isch/Actions/ISlOOl). The authors wish to thank Dr Giorgio Gambino for useful discussion on RNA interfer- ence, and Dr Sarah Luczaj and Prof Lukasz Luczaj for editing the manuscript.

References

References

1. International Service for the Acquisition of Agri-Biotech Ap- plications (ISAAA), Ithaca, NY, USA (http://www.isaaa.org/).

2. Food and Agriculture Organization of the United Nations (FAO), Rome, Italy (http://www.fao.org/).

3. International Centre for Genetic Engineering and Biotech- nology (ICGEB), Trieste, Italy (http://www.icgeb.orgfliome.html).

4. Joint Research Centre (JRC), European Commission, Brus- sels, Belgium (http://ec.europa.eu/dgs/jrc/index.cfm).

5. European Food Safety Authority (EFSA), Parma, Italy (http:// www.efsa.europa.eu/).

6. I. Metzler, A. Webster, Bio-objects and their boundaries: Governing matters at the intersection of society, politics, and science, Croat. Med. ]. 52 (2011) 648-650.

7. W. Parrot, Genetically modified myths and realities, New Biotechnol. 27 (2010) 545-551.

8. J.M. Dunwell: Intellectual Property Aspects of Plant Trans- formation. In: Plant Transformation Technologies, C.N. Stew- art, A. Touraev, V. Citovsky, T. Tzfira (Eds.), Wiley-Black- well, Hobokern, NJ, USA (2010) pp. 243-270.

9. M.P. Ferretti, V. Pavone, What do civil society organiza- tions expect from participation in science? Lessons from Germany and Spain on the issue of GMOs, Sei. Public Poli- cy, 36 (2009) 287-299.

10. L. Martineiii: Gene Transfer: Technical Visions and Emo- tional Perceptions. In: Psychology of Risk Perception, J.G. La- vino, R.B. Neumann (Eds.), Nova Science Publishers Inc., Hauppauge, NY, USA (2010) pp. 47-70.

11. M. Chrupek, H. Siipi, L. Martinelli, Bio-objects as 'bound- ary crawlers': The case of microRNAs, Croat. Med. ]. 53 (2012) 285-288.

12. L. Martinelli, M. Karbarz, H. Siipi, Science, safety and trust: The case of transgenic food, Croat. Med. ]. 54 (2013) 91-96.

13. A.J. Framond, M.W. Bevan, K.A. Barton, F. Flavell, M.D. Chilton, Mini-Ti plasmid and a chimeric gene construct: New approaches to plant gene vector construction, Advances in Gene Technology/: Molecular Genetics of Plants and Animals, Miami Winter Symposia, Vol. 20, K. Downey (Ed.), Academ- ic Press, New York, NY, USA (1983) pp. 159-170.

14. J. Schell, M. van Montagu, M. Holsters, P. Zambryski, H. Joos, D. Inze et al., Ti plasmids as experimental gene vec- tors for plants, Advances in Gene Technology: Molecular Gen- etics of Plants and Animals, Miami Winter Symposia, Vol. 20, K. Downey (Ed.), Academic Press, New York, NY, USA (1983) pp. 191-209.

15. R.T. Fraley, S.B. Rogers, R.B. Horsch, Use of a chimeric gene to confer antibiotic resistance to plant cells, Advances in Gene Technology: Molecular Genetics of Plants and Animals, Miami Winter Symposia, Vol. 20, K. Downey (Ed.), Academ- ic Press, New York, NY, USA (1983) pp. 211-221.

16. N. Murai, D.W. Sutton, M.G. Murray, J.L. Slightom, D.J. Merlo, N.A. Reichert et ah, Phaseolin gene from bean is ex- pressed after transfer to sunflower via tumor-inducing plas- mid vectors, Science, 222 (1983) 476-482.

17. J.K.C. Ma, E. Barros, R. Bock, P. Christou, P.J. Dale, P.J. Dix et ah, Molecular farming for new drugs and vaccines: Cur- rent perspectives on the production of pharmaceuticals in transgenic plants, EMBO Rep. 6 (2005) 593-599.

18. C. Conesa, M. Calvo, L. Sánchez, Recombinant human lacto- ferrin: A valuable protein for pharmaceutical products and functional foods, Biotechnol. Adv. 28 (2010) 831-838.

19. K. Yonekura-Sakakibara, K. Saito, Review: Genetically mo- dified plants for the promotion of human health, Biotech- nol. Lett. 28 (2006) 1983-1991.

20. T. Macek, P. Kotrba, A. Svatos, M. Nováková, K. Demne- rová, M. Macková, Novel roles for genetically modified plants in environmental protection, Trends Biotechnol. 26 (2008) 146-152.

21. P.G. Lemaux, Genetically engineered plants and foods: A scientist's analysis of the issues (Part I), Annu. Rev. Plant Biol. 59 (2008) 771-812.

22. P.G. Lemaux, Genetically engineered plants and foods: A scientist's analysis of the issues (Part II), Annu. Rev. Plant Biol. 60 (2009) 511-559.

23. R. Mittler, ?. Blumwald, Genetic engineering for modern agriculture: Challenges and perspectives, Annu. Rev. Plant Biol. 61 (2010) 443^62.

24. A. Harfouche, R. Meilan, A. Altman, Tree genetic engineer- ing and applications to sustainable forestry and biomass production, Trends Biotechnol. 29 (2011) 9-17.

25. V.M. Ursin, Modification of plant lipids for human health: Development of functional land-based omega-3 fatty acids, J. Nutr. 133 (2003) 4271^274.

26. S. Römer, P.D. Fraser, J.W. Kiano, C.A. Shipton, N. Misa- wa, W. Schuch, P.M. Bramley, Elevation of the pro-vitamin A content of transgenic tomato plants, Nat. Biotechnol. 18 (2000) 666-669.

27. C. Rosati, R. Aquilani, S. Dharmapuri, P. Pallara, C. Maru- sic, R. Tavazza et ah, Metabolic engineering of beta-caro- tene and lycopene content in tomato fruit, Plant ]. 24 (2000) 413^19.

28. G. Diretto, S. Al-Babili, R. Tavazza, V. Papacchioli, P. Beyer, G. Giuliano, Metabolic engineering of potato carotenoid content through tuber-specific overexpression of a bacter- ial mini-pathway, PLoS ONE, 2 (2007) e350.

29. BioCassava Plus, Donald Danforth Plant Science Center, St. Louis, MO, USA (http://nmrw.danforthcenter.org/science/programs/ international jprograms/bcp/).

30. Golden Rice is part of the solution, Golden Rice Humani- tarian Board, International Rice Research Institute (IRRI), Los Baños, Philippines (http://www.goldenrice.org/).

31. History of the Golden Rice Programme, Golden Rice Human- itarian Board, International Rice Research Institute (IRRI), Los Baños, Philippines (http://www.goldenrice.org/Contentl- -Who/who.php).

32. X. Ye, S. Al-Babili, A. Klöti, J. Zhang, P. Lucca, P. Beyer, I. Potrykus, Engineering the provitamin A (?-carotene) bio- synthetic pathway into (carotenoid-free) rice endosperm, Science, 287 (2000) 303-305.

33. P. Beyer, S. Al-Babili, X. Ye, P. Lucca, P. Schaub, R. Welsch, I. Potrykus, Golden Rice: Introducing the ß-carotene bio- synthesis pathway into rice endosperm by genetic engineer- ing to defeat vitamin A deficiency, /. Nutr. (Suppl.), 132 (2002) 506-510.

34. P. Schaub, S. Al-Babili, R. Drake, P. Beyer, Why is Golden Rice golden (yellow) instead of red?, Plant Physiol. 138 (2005) 441^50.

35. I. Potrykus, Golden Rice and beyond, Plant Physiol. 125 (2001) 1157-1161.

36. J.A. Paine, C.A. Shipton, S. Chaggar, R.M. Howells, M.J. Kennedy, G. Vernon et al., Improving the nutritional value of Golden Rice through increased pro-vitamin A content, Nat. Biotechnol. 23 (2005 ) 482^87.

37. G. Tang, J. Qin, G.G. Dolnikowski, R.M. Russell, M.A. Gru- sak, Golden Rice is an effective source of vitamin A, Am. ]. Clin. Nutr. 89 (2009) 1776-1783.

38. C.P. Nielsen, K. Anderson, Golden Rice and the looming GMO trade debate: Implication for the poor, Discussion Paper No. 0322, CIES, University of Adelaide, Adelaide, Australia (2003) pp. 1-45.

39. D. Dawe, R. Robertson, L. Unnevehr, Golden rice: What role could it play in alleviation of vitamin A deficiency?, Food Policy, 27 (2002) 541-560.

40. A.J. Stein, H.P.S. Sachdev, M. Qaim, Potential impact and cost-effectiveness of Golden Rice, Nat. Biotechnol. 24 (2006) 1200-1201.

41. M. Enserink, Tough lessons from Golden Rice, Science, 320 (2008) 468^71.

42. International Rice Research Institute (IRRI), Los Baños, Laguna, Philippines (http://irri.org/).

43. Grains of Delusion: Golden Rice Seen from the Ground, GRAIN, Barcelona, Spain (2001) (http://www.grain.org/article/ entries/10-grains-of-delusion-golden-rice-seen-from-the-ground).

44. Golden rice's lack of lustre: Addressing vitamin A deficien- cy without genetic engineering, Greenpeace International (2010) (www.greenpeace.org).

45. V. Shiva, The Golden rice' hoax - When public relations replaces science (http://online.sfsu.edu/~rone/GEessays/golden- ricehoax.html).

46. L. Bavaresco, C. Fregoni, E. Cantù, M. Trevisan: Stilbene compounds: From the grapevine to wine, Drugs Exp. Clin. Res. 25 (1999) 57-63.

47. P. Jeandet, A.C. Douillet-Breuil, R. Bessis, S. Debord, M. Sbaghi, M. Adrian, Phytoalexins from the Vitaceae: Biosynth- esis, phytoalexin gene expression in transgenic plants, anti- fungal activity, and metabolism, /. Agrie. Food Chem. 50 (2002) 2731-2741.

48. K. Das, Resveratrol in cardioprotection: A therapeutic pro- mise of alternative medicine, Mol. Interv. 6 (2006) 36^7.

49. P. Langcake, R.J. Pryce, The production of resveratrol by Vitis vinifera and other members of the Vitaceae as a re- sponse to infection or injury, Physiol. Plant Pathol. 9 (1976) 77-86.

50. P. Kopp, Resveratrol, a phytoestrogen found in red wine. A possible explanation for the conundrum of the 'French paradox'?, Eur. ]. Endocrinol. 138 (1998) 619-620.

51. S. Renaud, M. De Lorgeril, Wine, Alcohol, plateted and french paradox for coronary heart disease, Lancet, 339 (1992) 1523- 1526.

52. E.N. Frankel, A.L. Waterhouse, P.L. Teissedre, Principal phe- nolic phytochemicals in selected California wines and their antioxidant activity in inhibiting oxidation of human low- -density-lipoproteins, /. Agrie. Food Chem. 43 (1995) 890- 894.

53. A.J. Clifford, T.J. Powers, S.E. Ebeler, J.D. Ebeler, N.D. Bills, S.H. Hinrichs et ah, Delayed tumors onset in transgenic mice fed an amino acid diet supplemented with red wines so- lids, Am. J. Clin. Nutr. 64 (1996) 748-756.

54. J.B. Ruidavets, P.L. Teissedre, S. Carando, M. Ferrieres, J.C. Cabanis, Catechin in the mediteranean diet: Vegetable, fruit or wine?, Atherosclerosis, 153 (2000) 107-117.

55. C. Auger, B. Caporiccio, N. Landrault, P.L. Teissedre, C. Laurent, G. Cros et ah, Red wine phenolic compounds reduce plasma lipids and apolipoprotein B, and prevent early aortic atherosclerosis in hypercholesterolemic golden Syrian hamsters (Mesocricetus auratus), ]. Nutr. 132 (2002) 1207-1213.

56. A.A.E. Bertelli, Modulatory effect of resveratrol, a natural phytoalexin, on endothelial adhesion molecules and intra- cellular signal transduction, Pharm. Biol. 36 (1998) 44-52.

57. P. Vitaglione, S. Sforza, G. Galaverna, C. Ghidini, Ν. Ca- poraso, P.P. Vescovi et ah, Bioavailability of írans-resvera- trol from red wine in humans, Mol. Nutr. Food Res. 49 (2005 ) 495-504.

58. C. DeFrancesco, Scientific Journals Notified Following Re- search Misconduct Investigation, UConn Today, University of Connecticut, Mansfield, CT, USA (2012) (http://today.uconn. edu/blog/2012/01/scientific-journals-notified-following-research- misconduct-investigation/).

59. E. Callaway, In vino non Veritas? Red wine researcher im- plicated in misconduct case, Nature News Blog (2012) (http:// blogs. nature.com/news/2012/01/in-vino-non-veritas-red-wine-re- searcher-implicated-in-misconduct-case.html).

60. B. Delaunois, S. Cordelier, A. Conreux, C. Clément, P. Jean- det, Molecular engineering of resveratrol in plants, Plant Biotechnol. ]. 7 (2009) 2-12.

61. R. Hain, ?. Bieseler, G. Kindl, G. Schröder, R. Stocker, Ex- pression of a stilbene synthase gene in Nicotiana tabacum results in synthesis of the phytoalexin resveratrol, Plant Mol. Biol. 15 (1990) 325-335.

62. R. Hain, H.J. Reif, ?. Krause, R. Langebartels, H. Kindl, B. Vornam et ah, Disease resistance results from foreign phy- toalexin expression in a novel plant, Nature, 361 (1993) 153- 156.

63. J.D. Hipskind, N.L. Paiva, Constitutive accumulation of a resveratrol-glucoside in transgenic alfalfa increases resist- ance to Phoma medicaginis, Mol. Plant Microbe Interact. 13 (2000) 551-562.

64. B.D. Kineman, A. Au, N.L. Paiva, M.S. Kaiser, E.C. Brum- mer, D.F. Birt, Transgenic alfalfa that accumulates piceid (irans-resveratrol-3-O-ß-D-glucopyranoside) requires the pre- sence of ?-glucosidase to inhibit the formation of aberrant crypt foci in the colon of CF-1 mice, Nutr. Cancer, 58 (2007) 66-74.

65. B.D. Kineman, E.C. Brummer, N.L. Paiva, D.F. Birt, Res- veratrol from transgenic alfalfa for prevention of aberrant crypt foci in mice, Nutr. Cancer, 62 (2010) 351-361.

66. P. La, W. Cai, J. Zhang, F. Zhang, R. Fang, Function of res- veratrol derived from transgenic plant expressing resvera- trol synthase gene, Chinese Sei. Bull. 46 (2001) 1103-1107.

67. T.F. Chia, I. Ng, Production of resveratrol in transgenic red lettuce for chemoprevention of cancer and cardiovascular diseases, Agrobiotechnology and Plant Tissue Culture, Proceed- ings of the Asia-Pacific Conference on Plant Tissue Culture and Agrobiotechnology, Singapore, 2000, S.S. Bhojwani, W.Y. Soh (Eds.), Science Publishers, Enfield, NH, USA (2003) pp. 133- 145.

68. M.A. Flaishman, L. Schlizerman, Y. Cohen, Z. Kerem, L. Sivan, Expression of the health-beneficial stilbenes in trans- genic 'Spadona' pear (Pyrus communis), Acta Hortic. 671 (2005) 283-288.

69. I. Szankowski, K. Briviba, J. Fleschhut, J. Schönherr, H.J. Jacobsen, H. Kiesecker, Transformation of apple (Malus do- mestica Borkh.) with the stilbene synthase gene from grape- vine (Vitis vinifera L.) and a PGIP gene from kiwi (Actinidia deliciosa), Plant Cell Rep. 22 (2003) 141-149.

70. S. Rühmann, D. Treutter, S. Fritsche, K. Briviba, I. Szan- kowski, Piceid (resveratrol glucoside) synthesis in stilbene synthase transgenic apple fruit, /. Agrie. Food Chem. 54 (2006) 4633^640.

71. J.E. Thomzik, K. Stenzel, R. Stöcker, P.H. Schreier, R. Hain, D.J. Stahl, Synthesis of a grapevine phytoalexin in trans- genic tomatoes (Lycopersicon esculentum Mill.) conditions resistance against Phytophthora infestans, Physiol. Mol. Plant Pathol. 51 (1997) 265-278.

72. G. Leckband, H. Lörz, Transformation and expression of a stilbene synthase gene of Vitis vinifera L. in barley and wheat for increased fungal resistance, Theor. Appl. Genet. 96 (1998) 1004-1012.

73. S. Fettig, D. Hess, Expression of a chimeric stilbene syn- thase gene in transgenic wheat lines, Transgenic Res. 8 (1999) 179-189.

74. H. Liang, J. Zheng, X.Y. Duan, B.Q. Sheng, S.G. Jia, D.W. Wang et al., A transgenic wheat with a stilbene synthase gene resistant to powdery mildew obtained by biolistic meth- od, Chin. Sei. Bull. 45 (2000 ) 634-638.

75. P. Stark-Lorenzen, B. Nelke, G. Hänbler, H.P. Mühlbach, J.E. Thomzik, Transfer of a grapevine stilbene synthase gene to rice (Oryza sativa L.), Plant Cell Rep. 16 (1997) 668-673.

76. A. Giorcelli, F. Sparvoli, F. Mattivi, A. Tava, A. Balestrazzi, U. Vrhovsek et al., Expression of the stilbene synthase (StSy) gene from grapevine in transgenic white poplar results in high accumulation of the antioxidant resveratrol glucosides, Transgenic Res. 13 (2004) 203-214.

77. A. Richter, ?. de Kathen, G. de Lorenzo, K. Briviba, R. Hain, G. Ramsay et al., Transgenic peas (Pisum sativum) expres- sing polygalacturonase inhibiting protein from raspberry (Rubus idaeus) and stilbene synthase from grape (Vitis vini- fera), Plant Cell Rep. 25 (2006) 1166-1173.

78. A. Schwekendiek, O. Spring, A. Heyerick, ?. Pickel, N.T. Pitsch, F. Peschke, D. de Keukeleire, Constitutive expres- sion of a grapevine stilbene synthase gene in transgenic hop (Humulus lupulus L.) yields resveratrol and its deriva- tives in substantial quantities, /. Agrie. Food Chem. 55 (2007) 7002-7009.

79. L.P. Pan, S.L. Yu, C.J. Chen, H. Li, Y.L. Wu, H.H. Li, Clon- ing a peanut resveratrol synthase gene and its expression in purple sweet potato, Plant Cell Rep. 31 (2012) 121-131.

80. F. Medina-Bolivar, J. Condori, A.M. Rimando, J. Hubsten- berger, K. Shelton, S.F. O'Keefe et al., Production and se- cretion of resveratrol in hairy root cultures of peanut, Phy- tocliemistry, 68 (2007) 1992-2003.

81. B. Delaunois, S. Cordelier, A. Conreux, C. Clément, P. Jean- det, Molecular engineering of resveratrol in plants, Plant Biotechnol. ]. 7 (2009) 2-12.

82. Y. Wang, H. Chen, O. Yu, Metabolic engineering of resve- ratrol and other longevity boosting compounds, BioFactors, 36 (2010) 394-400.

83. D. Donnez, P. Jeandet, C. Clément, ?. Courot, Bioproduc- tion of resveratrol and stilbene derivatives by plant cells and microorganisms, Trends Biotechnol. 27 (2009) 706-713.

84. E. Fan, K. Zhang, M. Zhu, Q. Wang, Obtaining resveratrol: From chemical synthesis to biotechnological production, Mini-Rev. Org. Chem. 7 (2010 ) 4272-281.

85. R. Jaenisch, B. Mintz, Simian virus 40 DNA sequences in DNA of healthy adult mice derived from ρ reimplantation blastocysts injected with viral DNA, Proc. Natl. Acad. Sei. USA, 71 (1974) 1250-1254.

86. R.E. Hammer, V.G. Pursei, C.E. Rexroad, R.J. Wall, D.J. Bolt, K.M. Ebert et al., Production of transgenic rabbits, sheep, and pigs by microinjection, Nature, 315 (1985) 680-683.

87. K. Roschlau, P. Rommel, L. Andreewa, M. Zackel, D. Rosch- lau, B. Zackel et al., Gene transfer experiments in cattle, /. Reprod. Fértil. (Suppl.), 38 (1989) 153-160.

88. E. Gootwine, I. Barash, A. Bor, I. Dekel, A. Friedler, M. Heller et ah, Factors affecting success of embryo collection and transfer in a transgenic goat program, Theriogenology, 48 (1997) 485^99.

89. J.W. Gordon, F.H. Ruddle, Integration and stable germ line transformation of genes injected into mouse pronuclei, Science, 214 (1981) 1244-1246.

90. F. Park, Lentiviral vectors: Are they the future of animal transgenesis?, Physiol. Genomics, 31 (2007) 159-173.

91. R. Jaenisch, Germ line integration and Mendelian trans- mission of the exogenous Moloney leukemia virus, Proc. Natl. Acad. Sei. USA, 73 (1976) 1260-1264.

92. W.A. Kues, H. Niemann, Advances in farm animal trans- genesis, Pre v. Vet. Med. 102 (2011) 146-156.

93. C. Galli, I. Lagutina, A. Perota, S. Colleoni, R. Duchi, F. Lucchini, G. Lazzari, Somatic cell nuclear transfer and transgenesis in large animals: Current and future insights, Reprod. Dornest. Anim. (Suppl. 3), 47 (2012) 2-11.

94. K. Smith, C. Spadafora, Sperm-mediated gene transfer: Ap- plications and implications, BioEssays, 27 (2005) 551-562.

95. A. Gossler, T.C. Doetschman, H. Eistattaer, M. Katz, W. Schmidt, R. Kemler, Transgenesis by means of blastocyst derived embryonic stem cell lines, Proc. Natl. Acad. Sei. USA, 83 (1986) 9065-9069.

96. L. Lai, J.X. Kang, R. Li, J. Wang, W.T. Witt, H.Y. Yong et al, Generation of cloned transgenic pigs rich in omega-3 fatty acids, Nat. Biotechnol. 24 (2006) 435^36.

97. P.K. Magnus, F.A. Lali, Transgenic milk, Vet. World, 1 (2008) 319-320.

98. T.A. Hoagland, M. Julian, J.W. Riesen, D. Schrieber, W.L. Fodor, Transgenic pigs as animal model for xenogenic trans- plantation, Theriogenologi/, 47 (1997) 224.

99. HS. Chang, CH. Lin, YC. Chen, W.C.Y. Yu, Using siRNA technique to generate transgenic animals with spatiotem- poral and conditional gene knockdown, Am. ]. Pathol. 165 (2004) 1535-1541.

100. L.M. Houdebine, Production of pharmaceutical proteins by transgenic animals, Comp. Immunol. Microbiol. Infect. Dis. 32 (2009)107-121.

101. M.B. Wheeler, Agricultural applications for transgenic live- stock, Trends Biotechnol. 25 (2007) 204-210.

102. B. Grisart, F. Farnir, L. Karim, N. Cambisano, J.J. Kim, A. Kvasv et al., Genetic and functional confirmation of the causality of the DGAT1 K232A quantitative trait nucleo- tide in affecting milk yield and composition, Proc. Natl. Acad. Sei. USA, 101 (2004) 2398-2403.

103. M. Ron, D. Kliger, E. Feldmesser, ?. Seroussi, ?. Ezra, J.I. Weiler, Multiple quantitative trait locus analysis of bovine chromosome 6 in the Israeli Holstein population by a daugh- ter design, Genetics, 159 (2001) 727-735.

104. Online Mendelian Inheritance in Animals (OMIA), The Uni- versity of Sydney, Sydney, Australia (http://omia.angis.org. au/liome/).

105. D.M.T. Fessier, C.D. Navarrete, Meat is good to taboo. Dietary proscriptions as a product of the interaction of psychological mechanisms and social processes, /. Cognit. Cult. 3 (2003) 1^0.

106. J.E. Womak, Advances in livestock genomics: Opening the barn door, Genome Res. 15 (2005) 1699-1705.

107. C.G. Elsik, R.L. Tellam, K.C. Worley, R.A. Gibbs, D.M. Muz- ny, G.M. Weinstock et ah, Bovine genome sequencing and analysis consortium. The genome sequence of taurine cattle: A window to ruminant biology and evolution, Science, 324 (2009) 522-528.

108. V.G. Pursel, C.A. Pinkert, K.F. Miller, D.J. Bolt, R.G. Camp- bell, R.D. Palmiter et ah, Genetic engineering of livestock, Science, 254 (1989) 1281-1288.

109. N. Maclean, R.J. Laight, Transgenic fish: An evaluation of benefits and risks, Fish Fisher. 1 (2000) 146-172.

110. AquAdvantage® Fish, AquaBounty Technologies Inc, Walt- ham, MA, USA (2012) (http://www.aquabounty.com/products/ products-295.aspx).

111. J. Tang, Fishy business? AquaBounty Technologies, the FDA, and genetically modified foods. Case Studies in Ethics, The Kenan Institute for Ethics, Duke University, Durham, NC, USA (http://nmrw.duke.edu/web/kenanethics/CaseStudies/FDA.pdf).

112. S. Connor, Ready to eat: The first GM fish for the dinner table, The Independent (2012) (http://www.independent.co.uk/ news/science/ready-to-eat-the-first-gm-fish-for-the-dinner-table- 8430639.html).

113. G. Gaskell, A. Allansdottir, N. Allum, P. Castro, Y. Esmer, C. Fischler et ah, The 2010 Eurobarometer on the life sci- ences, Nat. Biotechnol. 29 (2011) 113-114.

114. European Patent Organization (EPO), The Hague, The Neth- erlands (http:// www.epo.org/).

115. ?. Liu, ?. Moav, A.J. Faras, K.S. Guise, A.R. Kapuscinski, P.B. Hackett, Development of expression vectors for trans- genic fish, Nat. Biotechnol. 8 (1990) 1268-1272.

116. S.J. Du, ?. Gong, G.L. Fletcher, Μ.A. Shears, M.J. King, D.R. Idler, C.L. Hew, Growth enhancement in transgenic Atlantic salmon by the use of an 'all fish' chimeric growth hormone gene construct, Nat. Biotechnol. 10 (1992) 176-181.

117. E. Jönsson, J.I. Johnsson, B.T. Björnsson, Growth hormone increases prédation exposure of rainbow trout, Proc. Roy. Soc. B, 263 (1996) 647-651.

118. L.F. Sundström, M. Löhmus, J.I. Johnsson, R.H. Devlin, Growth hormone transgenic salmon pay for growth po- tential with increased prédation mortality, Proc. Roy. Soc. ? (Suppl. 5), 271 (2004) 350-352.

119. E. Stokstad, Engineered fish: Friend or foe of the environ- ment?, Science, 297 (2002) 1797-1799.

120. V.G. Pursel, C.E. Rexroad Jr., Status of research with trans- genic farm animals, J. Anim. Sei. (Suppl. 3), 71 (1993) 10-19.

121. V.G. Pursel, R.J. Wall, M.B. Solomon, D.J. Bolt, J.D. Mur- ray, K.A. Ward, Transfer of ovine metallothionein-ovine growth hormone fusion gene into swine, /. Anim. Sei. 75 (1997) 2208-2214.

122. T.B. Mepham, R.D. Combes, M. Balls, O. Barbieri, H.J. Blok- huis, P. Costa et ah, The use of transgenic animals in the European Union: The Report and Recommendations of ECVAM Workshop Report No. 28, Altern. Lab. Anim. 26 (1998) 21^3.

123. C.E. Rexroad, R.E. Hammer, D.J. Bolt, K.E. Mayo, L.A. Frohman, R.D. Palmiter, R.L. Brinster, Production of trans- genic sheep with growth-regulating genes, Mol. Reprod. Dev. 1 (1989) 164-169.

124. C.E. Rexroad, K.E. Mayoa D.J. Bolt, T.H. Elsasser, K.F. Miller, R.R. Behringer et ah, Transferrin- and albumin-directed expression of growth-related peptides in transgenic sheep, J. Anim. Sei. 69 (1991) 2995-3004.

125. A. Bartke, M. Cecim, K. Tang, R.W. Steger, V. Chandras- hekar, D. Turyn, Neuroendocrine and reproductive conse- quences of overexpression of growth hormone in trans- genic mice, Exp. Biol. Med. 206 (1994) 345-359.

126. N.D. Riediger, R.A. Othman, M. Suh, M.H. Moghadasian, A systemic review of the roles of n-3 fatty acids in health and disease, /. Am. Diet. Assoc. 109 (2009) 668-679.

127. J.X. Kang, J. Wang, L. Wu, Z.B. Kang, Transgenic mice: Fat-1 mice convert n-6 to n-3 fatty acids, Nature, 427 (2004) 504.

128. X. Wu, H. Ouyang, B. Duan, D. Pang, L. Zhang, T. Yuan et ah, Production of cloned transgenic cow expressing omega- -3 fatty acids, Trans. Res. 21 (2012) 537-543.

129. Y. Echelard, C.A. Ziomek, H.M. Meade, Production of re- combinant therapeutic proteins in the milk of transgenic animals, BioPharm International (2006) (http://license.icopyright. net/user/viewFreeUse.act?fuid=MTYxMzQ5NjY%3D).

130. Food and Drug Administration (FDA), Silver Spring, MD, USA (www.fda.gov).

131. H. Baldassarre, B. Wang, C.L. Keefer, A. Lazaris, C.N. Cara- tzas, State of the art in the production of transgenic goats, Reprod. Fert. Develop. 16 (2004) 465^70.

132. M.B. Wheeler, G.T. Bleck, S.M. Donovan, Transgenic alter- ation of sow milk to improve piglet growth and health, Reprod. Suppl. 58 (2001) 313-324.

133. E.A. Maga, C.F. Shoemaker, J.D. Rowe, R.H. Bondurant, G.B. Anderson, J.D. Murray, Production and processing of milk from transgenic goats expressing human lysozyme in the mammary gland, J. Dairy Sei. 89 (2006) 518-24.

134. A. Haug, A.T. Hastmark, O.M. Harstad, Bovine milk in human nutrition - A review, Lipids Health Dis. 6 (2007) Article No. 25.

135. G. Laible, Enhancing livestock through genetic engineer- ing - Recent advances and future prospects, Comp. Immu- nol. Microbiol. Infect. Dis. 32 (2009) 123-137.

136. J.C. Mercier, Genetic engineering applied to milk-produc- ing animals: Some expectations, Exploiting New Technolo- gies in Animal Breeding, Genetic Developments, Proceedings of a Seminar in the CEC Animal Husbandry Research Programme, Oxford University Press, Oxford, UK (1987) pp. 122-131.

137. AgResearch, Hamilton, New Zealand (http://www.agresearch. co. nz/Pages/defa ult.aspx).

138. B. Brophy, G. Smolenski, T. Wheeler, D. Wells, P. L'Huil- lier, G. Laible, Cloned transgenic cattle produce milk with higher levels of ß-casein and κ-casein, Nat. Biotechnol. 21 (2003) 157-162.

139. R. Jenness: Composition of Milk. In: Fundamentals of Dairy Chemistry, AVI, New York, NY, USA (1995) pp. 1-38.

140. P.H. van Berkel, M.M. Welling, M. Geerts, H.A. van Veen, B. Ravensbergen, M. Salaheddine et ah, Large scale pro- duction of recombinant human lactoferrin in the milk of transgenic cows, Nat. Biotechnol. 20 (2002) 484^87.

141. E.A.J. Thomassen, H.A. van Veen, P.H.C. van Berkel, J.H. Nuijens, J.P. Abrahams, The protein structure of recombi- nant human lactoferrin produced in the milk of transgenic cows closely matches the structure of human milk-derived lactoferrin, Transgenic Res. 14 (2005) 397-405.

142. M.F. Brink, M.D. Bishop, F.R. Pieper, Developing efficient strategies for the generation of transgenic cattle which pro- duce biop harmaceuticals in milk, Theriogenology, 53 (2000) 139-148.

143. Naturalis Biodiversity Center, Leiden, The Netherlands (http://www.naturalis.nl/nl/).

144. R. Gray, Genetically modified cows produce 'human' milk, The Telegraph (2011) (http://nmrw.telegraph.co.uk/earth/agriculture/ geneticmodification/8423536/Genetically-modified-cows-produce- -human-milk.html).

145. National Agricultural Technology Institute (INTA), Buenos Aires, Argentina (http://inta.gob.ar/videos/rosita-isa-dio-leclie- -maternizada-nota-nicolas-mucci/view).

146. B.P. Bloomfield, B. Doolin, Imagination and technoscien- tific innovations: Governance of transgenic cows in New Zealand, Social Stud. Sei. 41 (2011) 59-83.

147. B.P. Bloomfield, B. Doolin, Symbolic communication in public protest over genetic modification: Visual rhetoric, symbolic excess, and social mores, Sei. Communie. (2013) (in press).

148. G.J. Hannon, F.V. Rivas, E.R Murchison, J.A Steitz, The expanding universe of noncoding RNAs, Cold Spring Har- bor Symposia On Quantitative Biology, Vol. 71, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (2006) pp. 551-564.

149. S. Ossowski, R. Schwab, D. Weigel, Gene silencing in plants using artificial microRNAs and other small RNAs, Plant ]. 53 (2008) 674-690.

150. F.F. de Felippes, J. Wang, D. Weigel, MIGS: mirRNA-in- duced gene silencing, Plant J. 70 (2012) 541-547.

151. S. Preuss, C.S. Pikaard: Targeted Gene Silencing in Plants Using RNA Interference. In: RNA Interference (RNAi): Nuts & Bolts of siRNA Technology, D. Engelke (Ed.), DNA Press, LLC, Collegeville, PA, USA (2003) pp. 23-36.

152. M.A. Gama Sosa, R. De Gasperi, G.A. Elder, Animal trans- genesis: An overview, Brain Struct. Fund. 214 (2010) 91- 109.

153. Y. Wu, J. Messing, RNA interference can rebalance the ni- trogen sink of maize seeds without losing hard endosperm, PLoS ONE, 7 (2012) e32850.

154. H.W. Dodo, K.N. Konan, FC. Chen, M. Egnin, O.M. Vi- quez, Alleviating peanut allergy using genetic engineering: The silencing of the immunodominant allergen Ara h 2 leads to its significant reduction and a decrease in peanut allergenicity, Plant Biotechnol. ]. 6 (2008) 135-145.

155. L.J.W.J. Gilissen, S.T.H.P. Bolhaar, C.I. Matos, G.J.A. Rou- wendal, M.J. Boone, F.A. Krens et ah, Silencing the major apple allergen Mal d 1 by using the RNA interference ap- proach, /. Allergy Clin. Immun. 115 (2005) 364-369.

156. B.N. Krath, F.D. Eriksen, B.H. Pedersen, L.J.W.J. Gilissen, W.E. van de Weg, L.O. Dragsted, Development of hypo- -allergenic apples: Silencing of the major allergen Mal d 1 gene in 'Elstar' apple and the effect of grafting, /. Hortic. Sei. Biotechnol. 84 (2009) 52-57.

157. E.M. Herman, R.M. Helm, R. Jung A.J. Kinney, Genetic modification removes an immunodominant allergen from soybean, Plant Physiol. 132 (2003) 36^3.

158. L.Q. Le, V. Mahler, Y. Lorenz, S. Scheurer, S. Biemelt, S. Vieths, U. Sonnewald, Reduced allergenicity of tomato fruits harvested from Lyc e 1-silenced transgenic tomato plants, /. Allergy Clin. Immunol. 118 (2006) 1176-1183.

159. G. Sunilkumar, L.M. Campbell, L. Puckhaber, R.D. Stipa- novic, K.S. Rathore, Engineering cottonseed for use in hu- man nutrition by tissue-specific reduction of toxic gossypol, Proc. Natl. Acad. Sei. USA, 103 (2006) 18054-18059.

160. A.C. McPherron, S.J. Lee, Double muscling in cattle due to mutations in the myostatin gene, Proc. Natl. Acad. Sei. USA, 94 (1997) 12457-12461.

161. J. Kota, C.R. Handy, A.M. Haidet, C.L. Montgomery, A. Eagle, L.R. Rodino-Klapac et ah, Follistatin gene delivery enhances muscle growth and strength in nonhuman pri- mates, Sei. Transi. Med. 1 (2009) 6ral5.

162. D. Zaborski, W. Grzesiak, I. Szatkowska, A. Dybus, M. Muszynska, M. Jedrzejczak, Factors affecting dystocia in cattle, Reprod. Dornest. Anim. 44 (2009) 540-551.

163. M.G. Stinnakre, J.L. Vilotte, S. Soulier, J.C. Mercier, Cre- ation and phenotypic analysis of alpha-lactalbumin defici- ent mice, Proc. Natl. Acad. Sei. USA, 91 (1994) 6544-6548.