Genetic Modification Of Animals Research Articles

The Broad Institute Scores Another Victory in Its Battle with the University of California over the Patenting of CRIPSR

The Broad Institute Scores Another Victory in Its Battle with the University of California over the Patenting of CRIPSR

Genetic Modification of Animals: Potential benefits and concerns

Genetic modification (GM), a process whereby gene and genotype frequencies are changed among individuals of each generation, is driven by natural and artificial forces. Natural forces include mutation, fitness and migration/introgression, while artificial forces include selection, crossbreeding and transgenesis/genetic engineering. Genetic modification, driven by natural forces, is essentially adaptive, while modification driven by artificial forces is controlled by human intervention aimed at meeting food, health and other needs. Conventional genetic modification, under sexual reproduction within species, produces both beneficial and negative effects. Modern genetic modification – interspecific exchange of genes using genetic engineering – has beneficial and negative effects as well, which are at varying degrees depending on the species involved. Control/management systems/mechanisms are developed and applied to enable societal benefits while minimizing/preventing negative effects of conventional and modern genetic modification. Targeted analysis of selected nutrients in animal products is made on a case-by-case basis to test substantial equivalence of any compositional changes resulting from genetic modification. Unique identifiers are established to track GM animals and their products in the food chain. 
 Modification génétique, processus par lequel les fréquences des gènes et des génotypes sont changes parmi les individus de chaque génération, est entraînée par des forces naturelles et artificielles. Les forces naturelles incluent la mutation, compétence de mère/père pour se reproduire/survivre et la migration / introgression. Les forces artificielles comprennent la sélection, le croisement et la transgénèse / génie génétique. La modification génétique entraînée par les forces naturelles est essentiellement adaptative, tandis que celle entraînée par les forces artificielles est contrôlée par une intervention humaine visant à répondre aux besoins alimentaires, sanitaires et autres. La modification génétique conventionnelle, lors de la reproduction sexuelle au sein des espèces, produit des effets à la fois bénéfiques et négatifs. Modification génétique moderne - échange interspécifique de gènes par génie génétique - a également des effets bénéfiques et négatifs mais à des degrés divers selon les espèces impliquées. Des systèmes / mécanismes de contrôle / gestion sont développés et appliqués pour permettre des avantages pour la société tout en minimisant / empêchant les effets négatifs des modifications génétiques conventionnelles et modernes. Une analyse ciblée de nutriments sélectionnés dans les produits d’origine animale est effectuée au cas par cas afin de tester l’équivalence substantielle de tout changement de composition résultant d’une modification génétique. Des identifiants uniques sont établis pour suivre les animaux GM et leurs produits dans la chaîne alimentaire.

Xenotransplantation of decellularized pig heart valves-Regulatory aspects in Europe.

The lack of human donors for allotransplantation forces the development of other strategies to circumvent the existing organ shortage documented on the waiting lists. Here, xenotransplantation offers a suitable option since the genetic modification of animals has become an established method that allows the generation of animals as donors of cells, tissues, and organs with reduced antigenicity. Focus is given on the generation of decellularized matrix scaffolds, for example, for valve transplantation and/or repair, that have the potential being fully assimilated by the recipient as they are no longer a mechanical implant with risk of calcification and related failure. This new class of products is transplants that will be regulated either as medical devices or as cell-based medicinal products, that is, advanced therapy medicinal products, according to the regulations in the European Union. In this review, we compile relevant regulatory aspects and point out the possibilities of how these products for human use may be regulated in the future.

Effect of ZFN-edited myostatin loss-of-function mutation on gut microbiota in Meishan pigs.

Intestine contains the body's second largest genetic information, so a relatively stable microbiota ecosystems and interactions between intestinal micro-organisms play a pivotal role in the normal growth and development in animals. The establishment of intestinal microflora is affected by a variety of factors such as species, environmental factors, developmental stage, organizational structure and physiological characteristics of various parts of the digestive tract. Gene editing technology such as ZFN has recently been used as a new approach to replace the traditional transgenic technology and to make genetic modifications in animals. However, it is not known if genetic modification by gene editing technology will have any impact on gut microbiota. In this study, by sequencing 16S rRNA collected from rectum, we investigated the effects of ZFN-mediated myostatin (MSTN) loss-of-function mutation (MSTN-/-) on gut microbiota in Meishan pigs. Our results showed that the fecal microbial composition is very similar between MSTN-/- Meishan pigs and wild type Meishan pigs. Although significant differences in certain individual strains were observed, all the dominant microorganism species are basically the same between MSTN-/- and wild type pigs. However, these differences do not adversely affect MSTN-/- Meishan pigs. Thus, it is concluded that ZFN-mediated MSTN loss-of-function mutation did not have any adverse effect on the gut microbiota in Meishan pigs.

CRISPR in Animals and Animal Models.

CRISPR-Cas9 has revolutionized the generation of transgenic animals. This system has demonstrated an unprecedented efficiency, multiplexability, and ease of use, thereby reducing the time and cost required for genome editing and enabling the production of animals with more extensive genetic modifications. It has also been shown to be applicable to a wide variety of animals, from early-branching metazoans to primates. Genome-wide screens in model organisms have been performed, accurate models of human diseases have been constructed, and potential therapies have been tested and validated in animal models. Several achievements in genetic modification of animals have been translated into products for the agricultural and pharmaceutical industries. Based on the remarkable progress to date, one may anticipate that in the future, CRISPR-Cas9 technology will enable additional far-reaching advances, including understanding the bases of diseases with complex genetic origins, engineering animals to produce organs for human transplantation, and genetically transforming entire populations of organisms to prevent the spread of disease.

Concepts and tools for gene editing.

Gene editing is a relatively recent concept in the molecular biology field. Traditional genetic modifications in animals relied on a classical toolbox that, aside from some technical improvements and additions, remained unchanged for many years. Classical methods involved direct delivery of DNA sequences into embryos or the use of embryonic stem cells for those few species (mice and rats) where it was possible to establish them. For livestock, the advent of somatic cell nuclear transfer platforms provided alternative, but technically challenging, approaches for the genetic alteration of loci at will. However, the entire landscape changed with the appearance of different classes of genome editors, from initial zinc finger nucleases, to transcription activator-like effector nucleases and, most recently, with the development of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas). Gene editing is currently achieved by CRISPR-Cas-mediated methods, and this technological advancement has boosted our capacity to generate almost any genetically altered animal that can be envisaged.

Impacts of genetically modified animals on the ecosystem and human activities

The genetic modification of animals to obtain transgenic animals started in 1980. The first transgenic animals were mice, which are still the most frequently used transgenic species. About 20 transgenic species have been obtained and they are more or less currently used. Various methods are being implemented to transfer foreign genes to the different species. Transgenic animals are mostly used for basic research to study gene and biological functions. Transgenic animals may also be relevant models to study human and animal diseases as well as to test new medicaments. Transgenics may also be the source of organs and cells for humans as well as of medicaments. The impact of transgenesis to improve animals for food and feed production is still non-existent but is expected to become a reality in the coming months. A few transgenic pets have been obtained and commercialized. The use of transgenic animals raises some welfare problems and specific regulation has been defined by the EU. Transgenesis in animals already has an impact on human activities and on the perception of living organisms by humans. The available techniques make the generation of transgenic humans plausible. This has not been achieved or proposed so far. The impact of transgenesis in humans might be huge. Theoretical and ethical discussions on this topic are underway.

Oxidative stress and redox regulation of gametogenesis, fertilization, and embryonic development.

Oxidative stress caused by elevated reactive oxygen species (ROS) is one of the predominant causes of both male and female infertility. Oxidative stress conditions cause either cell death or senescence by oxidation of cellular molecules including nucleic acid, proteins, and lipids. It is particularly important to minimize oxidative stress when in vitro fertilization is performed for the purpose of assisted reproduction. The problems associated with assisted reproductive technology are becoming evident, and it is now the time to clarify its mechanisms and cope with them. On the other hand, the beneficial roles of ROS, such as intracellular signaling, have become evident. The antithetical functions of ROS make it more difficult to overcome the problems caused by oxidative stress. Despite the difficulty in understanding mammalian reproduction, the mechanisms and problems can be gradually unveiled by advanced technology such as genetic modification of animals.

WHY DO WE NEED TRANSGENIC ANIMALS?

Genetic modification of animals through cross breeding and selection has probably been practiced since wild animals were first domesticated, but became more intensive beginning ~200 years ago with the creation of defined breeds with specialised phenotypes. This generality applies both to agriculturally important species, such as chickens and cattle, and to companion animals, such as cats and dogs. It also applies to plants. In the 20th century, new technologies designed to improve agricultural productivity, perhaps best illustrated by the development of artificial insemination and sperm cryopreservation for the dairy industry, as well as improved nutrition and veterinary care, all fueled by generous government support of agricultural research, accelerated progress towards providing low-cost, safe, and nutritious food for a burgeoning world population. Progress in some cases has been quite remarkable, especially in milk and meat production. The report by Palmiter, Brinster and colleagues in 1982, that mice harboring a transgene for growth hormone under the control of a metallothionein gene promoter grew faster than control siblings, raised the spectre of using relatively simple, one-step gene insertions, rather than cross-breeding and selection, to improve the production of milk, meat, and fibre, and enhance animal health quickly and directly. Although agronomy and horticulture have been able to benefit greatly from applying genetic modification to plants, there has been only very slow application of transgenesis to animal agriculture and aquaculture. The main goal of my talk will be to illustrate some of the difficulties, both scientific and social, that have been encountered in bringing genetic modification and related technologies, such as cloning, into mainstream agriculture. I shall also discuss the competitive advantage held by rodents over alternative animal models in biomedical sciences but argue that the latter still have an important role to play in this regard. A final topic will be to review the potential value of pluripotent stem cells for introducing genetic change in domestic species and for testing the efficacy and safety of grafts derived from pluripotent stem cells before human clinical trials.

56 ESTABLISHMENT OF TRANSGENIC RED FLUORESCENCE PROTEIN (RFP) CLONE DOGS THROUGH A STABLE TRANSMISSION OF RFP GENE TO NEXT GENERATION

Somatic cell nuclear transfer (SCNT) technology has been spotlighted not only for its advantage in producing unlimited numbers of genetically identical animals, but also the possibility of producing complex genetic modifications in animals. However, a few reports showed that mosaic expression of transgene in transgenic animals produced by SCNT (Park et al. 2002) and down-regulated gene expression is sometimes irreversible in their offspring (Bordignon et al. 2003). Therefore, we investigated reproductive ability by a breeding between female transgenic beagles and wild-type beagles. When female transgenic beagles (R1, R2, R3, and R5) expressing red fluorescence protein (RFP) gene reached puberty at 373, 353, 283, and 354 days after birth, serum progesterone concentration was monitored for detecting timing of ovulation. Approximately 72 to 79 h after ovulation, the beagles were naturally mated or artificially inseminated. Pregnancy was confirmed by ultrasonography at Day 30 after insemination. The transgenic bitches (R1, R2, R3, and R5) were then bred with wild-type male dogs, became pregnant, and successfully delivered 13 puppies (9 female and 4 male). In order to prove integration of RFP gene in all offspring, DNA was extracted from the blood of pups on Day 7 after birth. For PCR analysis, a primer pair for the RFP gene, forward primer (5′CGTGAAGCTGAAGGTGA-3′) and reverse primer (5′-CTCGTACTGCTCCACGA-3′), were used to amplify a 517-bp DNA fragment. The initial denaturation was performed at 94°C for 5 min, followed by 30 cycles at 94°C for 40 s (denaturation), 58°C for 40 s (annealing), and 72°C for 40 s (extension), and a final incubation at 72°C for 10 min to ensure complete strand extension. Presence of the RFP transgene in 7 of the puppies was confirmed by PCR and the puppies expressed RFP upon UV illumination. It was not different from the 53.8% expected Mendelian ratio. The present result demonstrated a stable transmission of the RFP gene into 5 female and 2 male offspring in the second generation. Among the second generation, 2 female puppies integrated with the RFP gene were in heat at ∼1-year-old. They were then bred with the semen of a wild-type beagle and bore 6 puppies. In the third generation, 3 puppies carried the RFP gene and results showed the expected Mendelian ratio. In conclusion, the present study demonstrates that female transgenic beagles have normal reproductive ability and a stable insertion of the transgene to the next generation. This study was financially supported by NRF (#M10625030005-508-10N25), SNU foundation (Benefactor; RNL BIO), BK 21 for Veterinary Science, and Purina Korea.

Production of Prnp −/− goats by gene targeting in adult fibroblasts

Homozygous mice devoid of functional Prnp are resistant to scrapie and prion propagation, but heterozygous mice for Prnp disruption still suffer from prion disease and prion deposition. We have previously generated heterozygous cloned goats with one allele of Prnp functional disruption. To obtain goats with both alleles of Prnp be disrupted which would be resistant to scrapie completely, a second-round gene targeting was applied to disrupt the wild type allele of Prnp in the heterozygous goats. By second-round gene targeting, we successfully disrupted the wild type allele of Prnp in primary Prnp (+/-) goat skin fibroblasts and obtained a Prnp (-/-) cell line without Prnp expression. This is the first report on successful targeting modification in primary adult somatic cells of animals. These cells were used as nuclear donors for somatic cell cloning to produce Prnp (-/-) goats. A total of 57 morulae or blastocytes developed from the reconstructed embryos were transferred to 31 recipients, which produced 7 pregnancies at day 35. At 73 days of gestation, we obtained one cloned fetus with Prnp (-/-) genotype. Our research not only indicated that multiple genetic modifications could be accomplished by multi-round gene targeting in primary somatic cells, but also provided strong evidence that gene targeting in adult cells other than fetal cells could be applied to introduce precise genetic modifications in animals without destroying the embryos.

‘Ethical concepts regarding the genetic engineering of laboratory animals’

Intrinsic value and animal integrity are two key concepts in the debate on the ethics of the genetic engineering of laboratory animals. These concepts have, on the one hand, a theoretical origin and are, on the other hand, based on the moral beliefs of people not directly involved in the genetic modification of animals. This 'external' origin raises the question whether these concepts need to be adjusted or extended when confronted with the moral experiences and opinions of people directly involved in the creation or use of transgenic laboratory animals. To answer this question, 35 persons from the practice of biomedical research who are directly involved in genetic engineering (scientists, biotechnicians, animal caretakers and laboratory animal scientists) were interviewed. They were asked to give their moral opinion on different aspects of the genetic engineering of animals and to react to statements about the concepts of intrinsic value and animal integrity. Analysis of the interviews showed that, contrary to what is often assumed, the respondents embraced these concepts, even those senses that (more) specifically apply to genetic engineering. And although the respondents raised some objections that go beyond issues of animal welfare, these objections could quite well be expressed in terms of the concepts of intrinsic value and animal integrity. In short, the results of the present study strongly suggest that these concepts do not have to be adjusted or extended in the light of the moral experiences and opinions from practice.

What do individuals in different science groups within a life sciences organization think about genetic modification?

An assessment was undertaken of the attitudes of individuals within the science community towards a program to produce genetically modified cattle for altered milk composition, expectantly allowing for research into the treatment of multiple sclerosis in humans. The majority of respondents to an electronic survey expressed favorable attitudes to the program, thought it beneficial, respected individual freedom and was fair and just and disagreed that it was harmful. A passion for science and having a suitable lifestyle were the most important motivating factors for individuals. Finally, there were a wide range of responses to a number of cultural beliefs or myths. Science grouping significantly affected the responses. Compared with Systems and Land groups, Plant and Reproduction groups more strongly agreed with the project, thought it less harmful to interest groups, felt that genetic modification of animals was more morally acceptable, and more strongly agreed with the myth statements. These results indicate a diversity of beliefs and attitudes towards genetic modification amongst those within the science community, and highlight the importance of understanding ethics and myths in dealing with them. It is suggested that the diversity of beliefs could be better used to help shape public policy and understanding of biotechnology.

Importance of fear in the case of genetically modified food

AbstractFear appeals concerning genetically modified food (GMF) frequently appear in the mass media. They have played a crucial role in creating widespread fear of GMF (also known as “Frankenstein food” among the fearful) in a large part of the world. The present study validates a scale to measure consumers' fear of GMF and shows that Dutch consumers feel significantly more fearful of GMF than of other new food types. There are no strong relations between consumers' sociodemographic makeup and fear of GMF, indicating that fear of this technologically new type of food is an emotion that cuts across society. Fear of GMF is positively influenced by consumers' concern for the environment and negatively affected by their faith in technology in food production. Consumers who are more fearful of GMF have a more negative attitude toward genetically modified food and toward genetic modification of animals, and exhibit a greater interest in information related to food production. © 2004 Wiley Periodicals, Inc.

Practical Difficulties in Balancing Harms and Benefits in the Modern Use of Laboratory Animals: Biotechnology

Every ethical review process presupposes a set of ethical criteria, proper procedures and a consistent legal framework. The Netherlands became unique in 1996, by the decision of the Parliament to start a national ethical review procedure regarding the ethical aspects of genetic modification of animals (Animal and Biotechnology Decree). Due to the policy of public disclosure of all relevant documents and providing a legal trajectory for public objections, extensive ethical arguments pro and con are well documented. This paper lists and analyses the misconceptions when one tries to apply the "classical" ethical framework of harms and benefits to the new designs of animal experiments in the context of biotechnology. For instance, what is the object? The foster mothers, the manipulated egg cell, the malformed embryo, the pups dying before weaning or the adult mice in the experiments? The public considers the whole methodology ethically relevant. Most researchers tend to check out only "consciously" experienced harm, which implies only autonomously living mice and not a cell or an embryo. And, what is the domain of benefits? An exciting construct to be incorporated in the genome, the healthy birth of a surviving knock out, or a successful experiment with adult genetically modified mice? In the same way, the question for relevant alternatives in the "classic" sense of the Three Rs seems to shift completely to a discussion about goals in the far future. Is this modification absolutely necessary, or are the goals also reached by research strategies without genetic modification?

Naturalness and the genetic modification of animals

In the past few years it has been recognised that so-called intrinsic concerns about genetic modification (GM) of plants and animals, for food in particular, have an important role in the public perception of GM. One of these concerns is the view that GM is ‘unnatural’. This article gives an overview of the often conflicting views on the argument of unnaturalness in books and reports. The author gives a new direction to this discussion, by contrasting the common sense view of nature and animals, with the scientific concept of nature and what is natural. The view of nature and what is natural is always normative. This is illustrated by making explicit the concept of nature in organic farming, which explains why GM is rejected.

Genetic modification of animals in the next century

Since the initial demonstration in 1982 of profound phenotypic effects stemming from the expression of a single transgene, genetic engineering has revolutionized fundamental biological and biomedical research. The application of transgenic technology to farm animals has held the promise of being able to improve animal agriculture significantly and has resulted in a new industry, i.e., the successful expression of foreign proteins in the mammary gland for the pharmaceutical industry. Work over the last few years in model species (e.g., the mouse) and new technical developments such as cloning have now set the stage for the initial application of transgenic technology for the improvement of farm animals. Major limitations that remain are the lack understanding of which genes we should transfer in order to alter quantitative production traits usefully and the low efficiency of producting transgenic founders. Furthermore, more research is needed concerning the consequences and potential problems arising from the integration of transgenes into populations with varying genetic backgrounds. Recent advances suggest that within the first decade of the 21 st century the first transgenic animals will become available to the livestock industry, with acceptance depending upon their cost versus their potential economic benefit to the producers.

Ethical Aspects of Genetic Modification of Animals: Opinion of the Group of Advisers on the Ethical Implications of Biotechnology of the European Commission

Ethical Aspects of Genetic Modification of Animals: Opinion of the Group of Advisers on the Ethical Implications of Biotechnology of the European Commission

Editorial

The first contribution in this issue of Animal Welfare is the second of the newly instigated Invited Essays, written at the invitation of the editor and aimed at addressing contemporary welfare problems. It is by Robert Hubrecht, Assistant Director of UFAW and it examines the techniques involved in the genetic modification of animals, and considers the welfare implications and the relevance of the current UK legislation. These essays, which will be an occasional feature of the journal, are refereed by independent experts but very much represent the personal views of the authors. Hopefully some of the opinions expressed will stimulate readers to respond with constructive critical comments, which could be published in the journal's Letters section. The original author will of course have the right to reply.