A brief overview of transgenic farm animals

Transgenesis offers new possibilities to rapidly modify the genome of living organisms. The application of transgenesis to farm animals faces many problems, more than those observed in the transgenesis of laboratory animals, as there are currently many different techniques available to obtain transgenic animals, which all have problems regarding low efficiency and high costs. When these techniques are applied to farm animals the problems concerning transgenesis are multiplied. Two main techniques, male pronuclear microinjection and sperm mediated gene transfer, utilised in farm animal transgenesis, are briefly presented. The improvement of these techniques and the employment of other biotechnologies such as cloning, could expand the uses of transgenic farm animals for human health.     

Transgenesis may affect farm animal welfare: a case for systematic risk assessment

This paper considers (potentially) harmful consequences of transgenesis for farm animal welfare and examines the strategy of studying health and welfare of transgenic farm animals. Evidence is discussed showing that treatments imposed in the context of farm animal transgenesis are by no means biologically neutral and may compromise animal health and welfare. Factors posing a risk for the welfare of transgenic farm animals include integration of a transgene within an endogenous gene with possible loss of host gene function (insertional mutations), inappropriate transgene expression and exposure of the host to biologically active transgene-derived proteins, and in vitro reproductive technologies employed in the process of generating transgenic farm animals that may result in an increased incidence of difficult parturition and fetal and neonatal losses and the development of unusually large or otherwise abnormal offspring (large offspring syndrome). Critical components of a scheme for evaluating welfare of transgenic farm animals are identified, related to specific characteristics of transgenic animals and to factors that may interact with the effects of transgenesis. The feasibility of an evaluation of welfare of transgenic farm animals in practice is addressed against the background of the objectives and conditions of three successive stages in a long-term transgenic program. Concrete steps with regard to breeding and testing of transgenic farm animals are presented, considering three technologies to generate transgenic founders: microinjection, electroporation and nuclear transfer, and gene targeting including gene knockout. The proposed steps allow for unbiased estimations of the essential treatment effects, including hemi- and homozygous transgene effects as well as effects of in vitro reproductive technologies. It is suggested that the implementation of appropriate breeding and testing procedures should be accompanied by the use of a comprehensive welfare protocol, specifying which parameters to monitor, at which stages of the life of a farm animal, and in how many animals. Some prerequisites and ideas for such a protocol are given. It is anticipated that systematic research into the welfare of farm animals involved in transgenesis will facilitate the use of the safest experimental protocols as well as the selection and propagation of the healthiest animals and, thereby, enable technological progress that could be ethically justified.     

Advances in farm animal transgenesis

The first transgenic livestock were produced in 1985 by microinjection of foreign DNA into zygotic pronuclei. This was the method of choice for more than 20 years, but more efficient protocols are now available, including somatic cell nuclear transfer and lentiviral transgenesis. Typical applications include carcass composition, lactational performance and wool production, as well as enhanced disease resistance and reduced environmental impact. Transgenic farm animal production for biomedical applications has found broad acceptance. In 2006 the European Medicines Agency (EMA) approved commercialization of the first recombinant pharmaceutical protein, antithrombin, produced in the mammary gland of transgenic goats. As the genome sequencing projects for various farm animal species are completed, it has become feasible to perform precise genetic modifications employing the emerging tools of lentiviral vectors, small interfering ribonucleic acids, meganucleases, zinc finger nucleases and transposons. We anticipate that genetic modification of farm animals will be instrumental in meeting global challenges in agricultural production and will open new horizons in biomedicine.     

转基因动物的制备方法及应用评述

近些年来转基因技术在动物领域取得了很大进展,作者主要对转基因动物的制备方法及其优缺点进行了综述,指出了转基因动物在畜牧业、医药产业和人类疾病模型的建立等方面的应用及其存在的问题,最后就其未来的发展进行了展望。     

转基因技术在畜牧兽医中的发展前景

通过简要叙述转基因动物研究的发展简史、研究现状和存在问题 ,对转基因动物育种、药物生物反应器、组织器官移植 ,以及基因治疗和免疫等前景进行了综述。在此基础上 ,从畜牧兽医角度出发提出了追踪发展趋势的几点思考。     

转基因在动物生产中的应用

人们一直期望畜产品和人类健康产品能快速得到提高,DNA重组技术和转基因的出现使得在不同物种间,甚至不同系统发育领域间的这一提高在很大范围内变为可能。如今,我们能在细菌中生产人类胰岛素,在牛奶中获得人类凝结因子。转基因、动物克隆和动物多产技术的进步在一定程度上已经实现了在动物转基因领域的期望。作者回顾了当前动物转基因乳、肉和转基因抗病动物的研究近况,并讨论了一些由转基因技术应用而引发的生物伦理学和商业性问题。     

转基因疾病动物模型的研究进展

人类疾病动物模型(animalmodelsofhumandiseases)是为阐明人类疾病的发生机制或建立治疗方法而制作的、具有人类疾病模拟表现的实验动物。疾病动物模型对医学发展做出了很大贡献。但是,许多疾病难以用人工诱发的方法制造动物模型,或许多疾病在实验动物身上不发生或仅仅是高等哺乳类动物才发生,因此难以通过自发或人工定向培育的方法获得动物模型。转基因技术的出现,为人类精确地研究基因与疾病的相关关系提供了可能,而且可以在个体发生的每个阶段中使用任何个体进行遗传功能的分析。因此,转基因疾病动物模型的开发成为转基因动物的热点。文章就转基因疾病动物模型的建立制作及应用前景做一综述。     

Use of intracytoplasmic sperm injection (ICSI) to generate transgenic animals

Even though intracytoplasmic sperm injection (ICSI) has been widely used for the production of offspring in human infertility clinics and in reproductive research laboratories using mice, many researchers engaged in animal transgenesis still consider it somewhat cumbersome. The greatest advantage of ICSI-mediated transgenesis is that it allows introduction of very large DNA transgenes (e.g., yeast artificial chromosomes), with relatively high efficiency into the genomes of hosts, as compared to pronuclear injection. Recently, we have developed an active form of intracytoplasmic sperm injection-mediated transgenesis (ICSI-Tr) with fresh sperm utilizing transposons. The transgenic efficiencies rival all transgenic techniques except that of lentiviral methods.     

Production of pharmaceutical proteins by transgenic animals

Proteins started being used as pharmaceuticals in the 1920s with insulin extracted from pig pancreas. In the early 1980s, human insulin was prepared in recombinant bacteria and it is now used by all patients suffering from diabetes. Several other proteins and particularly human growth hormone are also prepared from bacteria. This success was limited by the fact that bacteria cannot synthesize complex proteins such as monoclonal antibodies or coagulation blood factors which must be matured by post-translational modifications to be active or stable in vivo. These modifications include mainly folding, cleavage, subunit association, gamma-carboxylation and glycosylation. They can be fully achieved only in mammalian cells which can be cultured in fermentors at an industrial scale or used in living animals. Several transgenic animal species can produce recombinant proteins but presently two systems started being implemented. The first is milk from farm transgenic mammals which has been studied for 20 years and which allowed a protein, human antithrombin III, to receive the agreement from EMEA (European Agency for the Evaluation of Medicinal Products) to be put on the market in 2006. The second system is chicken egg white which recently became more attractive after essential improvement of the methods used to generate transgenic birds. Two monoclonal antibodies and human interferon-beta 1a could be recovered from chicken egg white. A broad variety of recombinant proteins were produced experimentally by these systems and a few others. This includes monoclonal antibodies, vaccines, blood factors, hormones, growth factors, cytokines, enzymes, milk proteins, collagen, fibrinogen and others. Although these tools have not yet been optimized and are still being improved, a new era in the production of recombinant pharmaceutical proteins was initiated in 1987 and became a reality in 2006. In the present review, the efficiency of the different animal systems to produce pharmaceutical proteins are described and compared to others including plants and micro-organisms.     

基因枪转化技术在动物转基因中应用的研究进展

基因枪转化技术无论在动物还是植物方面都产生了巨大的影响,因粒子介导的转基因方法简单、安全、高效,而越来越被人们所重视。研究结果表明,该转化技术不仅能够用于组织、细胞、器官,甚至可用于一些较难转染的靶目标,同时还可用于体内和体外信息的转换。目前手提式基因枪已广泛应用于皮肤、组织、各种细胞或内脏器官的转染,应用最广的领域是基因治疗,主要包括基因免疫和抑制肿瘤生长。近几年,基因枪技术发展迅速,应用范围越来越广,已经取得了丰硕的成果。作者主要叙述了基因枪转化的基本原理及近年来人们对基因枪转化技术条件的改进,同时就基因枪在动物转基因中的应用和一些主要的研究成果作了阐述,主要包括利用基因枪基因免疫、基因治疗、自杀基因治疗癌症、胚胎转基因、各种动物细胞和器官组织转基因等,并对基因枪技术在动物上应用的优势和不足进行了一些分析,最后展望了基因枪转化技术在动物中的应用前景。     

Establishing a laboratory animal model from a transgenic animal: RasH2 mice as a model for carcinogenicity studies in regulatory science

Transgenic animal models have been used in small numbers in gene function studies in vivo for a period of time, but more recently, the use of a single transgenic animal model has been approved as a second species, 6-month alternative (to the routine 2-year, 2-animal model) used in short-term carcinogenicity studies for generating regulatory application data of new drugs. This article addresses many of the issues associated with the creation and use of one of these transgenic models, the rasH2 mouse, for regulatory science. The discussion includes strategies for mass producing mice with the same stable phenotype, including constructing the transgene, choosing a founder mouse, and controlling both the transgene and background genes; strategies for developing the model for regulatory science, including measurements of carcinogen susceptibility, stability of a large-scale production system, and monitoring for uniform carcinogenicity responses; and finally, efficient use of the transgenic animal model on study. Approximately 20% of mouse carcinogenicity studies for new drug applications in the United States currently use transgenic models, typically the rasH2 mouse. The rasH2 mouse could contribute to animal welfare by reducing the numbers of animals used as well as reducing the cost of carcinogenicity studies. A better understanding of the advantages and disadvantages of the transgenic rasH2 mouse will result in greater and more efficient use of this animal model in the future.     

Production of transgenic livestock: promise fulfilled

The introduction of specific genes into the genome of farm animals and its stable incorporation into the germ line has been a major technological advance in agriculture. Transgenic technology provides a method to rapidly introduce "new" genes into cattle, swine, sheep, and goats without crossbreeding. It is a more extreme methodology, but in essence, not really different from crossbreeding or genetic selection in its result. Methods to produce transgenic animals have been available for more than 20 yr, yet recently lines of transgenic livestock have been developed that have the potential to improve animal agriculture and benefit producers and/or consumers. There are a number of methods that can be used to produce transgenic animals. However, the primary method to date has been the microinjection of genes into the pronuclei of zygotes. This method is one of an array of rapidly developing transgenic methodologies. Another method that has enjoyed recent success is that of nuclear transfer or "cloning." The use of this technique to produce transgenic livestock will profoundly affect the use of transgenic technology in livestock production. Cell-based, nuclear transfer or cloning strategies have several distinct advantages for use in the production of transgenic livestock that cannot be attained using pronuclear injection of DNA. Practical applications of transgenesis in livestock production include enhanced prolificacy and reproductive performance, increased feed utilization and growth rate, improved carcass composition, improved milk production and/or composition, and increased disease resistance. One practical application of transgenics in swine production is to improve milk production and/or composition. To address the problem of low milk production, transgenic swine over-expressing the milk protein bovine alpha-lactalbumin were developed and characterized. The outcomes assessed were milk composition, milk yield, and piglet growth. Our results indicate that transgenic overexpression of milk proteins may provide a means to improve swine lactation performance.     

Characterization of transgenic livestock production

The objective of transgenic livestock improvement projects is to develop and bring to market superior breeding stock, as well as germplasm for the artificial insemination and embryo transfer industries. Livestock animal biotechnology programs hold the promise of achieving, in a single generation, improvements in commercially important livestock species previously possible only through long-term traditional selective breeding practices or by chance mutation. Transgenic farm animals harboring growth hormone or metabolically related structural genes have been created. Studies of these animals demonstrate the effects of inadequate regulation of transgene expression. Research continues to explore the intricacies of developmental regulation of such genes and phenotypic consequences of mammalian gene transfer. Ultimately, genetically engineered livestock will provide producers with the benefit of increased production efficiencies while the consumer will have healthier animal food products. Conceivably, products will be produced with lower levels of fat, cholesterol, feed additives and pharmaceutical residues from animals with altered carcass composition that will result in greater nutritional benefit for the consumer.     

转基因动物在免疫学研究中的应用

转基因动物技术在分子免疫、免疫耐受、自身免疫、移植免疫、抗感染免疫和免疫防治等研究中的应用日益广泛。表达功能性重排的 T细胞抗原受体 (TCR)的转基因动物的应用加深了人们对 TCR基因重排、T细胞在胸腺中的发育过程、αβT细胞和γδT细胞分化途径及 T细胞免疫耐受形成机制的认识 ;免疫分子及其受体转基因动物成为研究其功能的直接手段 ;病毒及病毒受体转基因动物为研究病毒致病机制、抗病毒治疗及疫苗评价提供了新的模型 ;自身免疫病和用于移植免疫研究的转基因动物模型成为探讨疾病发病机制及治疗手段的有力工具。目前利用转基因动物已生产出多种抗体、干扰素、IL-2、TNFα、G-CSF、L F等诊断和治疗性生物活性蛋白     

Mammary expression of new genes to combat mastitis

Continual advances in the ability to produce transgenic animals make it likely that such animals will become important components of animal agriculture. The full benefit of the technology, and justification of its initial cost outlay, will be dependent on the establishment within these animals of new traits not easily achievable by other means. Potential applications include enhanced nutrient digestibility with reduced fecal losses, significantly altered milk composition with superior nutritional properties, and enhanced disease resistance. Our goal is to enhance mastitis resistance of dairy cows by enabling the cells of the mammary gland to secrete additional antibacterial proteins. Proof of concept has been obtained through experimentation with a transgenic mouse model. Three lines of mice were developed that produce varying levels of lysostaphin in their milk. This protein has potent anti-staphylococcal activity and its secretion into milk confers substantial resistance to infection caused by intramammary challenge with Staphylococcus aureus, a major mastitis pathogen. Additional antibacterial proteins are being sought that will complement lysostaphin. A potential benefit of transgenic application of antibacterial proteins is the concomitant sparing in the agricultural use of antibiotics currently used as human therapeutics. Antibacterial proteins, such as lysostaphin, are not typically used as injectable or oral therapeutics because of immune-mediated or digestive destruction of their activity. In contrast, the immune system of transgenic animals will not consider the transgenic protein as being foreign. In addition we are exploring the potential of involution or mastitis responsive promoter elements for use in subsequent transgenic experiments designed to restrict lysostaphin production to these important time points. It is anticipated that genomics will play a role in unveiling candidate genes whose promoter elements will enable desired temporal expression patterns. The transgenic approach to insertion of new genetic material into agriculturally important animals is feasible but requires extensive prior evaluation of the transgene and transgene product in model systems.     

乳蛋白基因在转基因动物乳腺细胞中特异性表达的研究进展

乳蛋白是乳的主要营养成分，乳蛋白的种类及其在乳中的浓度都影响乳的品质，而乳中各乳蛋白的含量都受到相应乳蛋白基因的控制。通过转基因技术，可以在转基因动物乳腺细胞中特异性地表达目的蛋白，从而改善乳的品质以及生产具有特殊药用价值的乳产品。本文主要概述乳蛋白多态性及其作用，乳蛋白基因及多态性，并着重介绍乳蛋白基因在转基因动物体内的表达调控；通过介绍大量转基因试验的研究成果，以探寻改变乳成分的分子遗传基础。     

Transgenic mammalian species, generated by somatic cell cloning, in biomedicine, biopharmaceutical industry and human nutrition/dietetics--recent achievements

Somatic cell cloning technology in mammals promotes the multiplication of productively-valuable genetically engineered individuals, and consequently allows also for standardization of transgenic farm animal-derived products, which, in the context of market requirements, will have growing significance. Gene farming is one of the most promising areas in modern biotechnology. The use of live bioreactors for the expression of human genes in the lactating mammary gland of transgenic animals seems to be the most cost-effective method for the production/processing of valuable recombinant therapeutic proteins. Among the transgenic farm livestock species used so far, cattle, goats, sheep, pigs and rabbits are useful candidates for the expression of tens to hundreds of grams of genetically-engineered proteins or xenogeneic biopreparations in the milk. At the beginning of the new millennium, a revolution in the treatment of disease is taking shape due to the emergence of new therapies based on recombinant human proteins. The ever-growing demand for such pharmaceutical or nutriceutical proteins is an important driving force for the development of safe and large-scale production platforms. The aim of this paper is to present an overall survey of the state of the art in investigations which provide the current knowledge for deciphering the possibilities of practical application of the transgenic mammalian species generated by somatic cell cloning in biomedicine, the biopharmaceutical industry, human nutrition/dietetics and agriculture.     

The role of genetic engineering in livestock production

In this paper we discuss the use of genetic engineering in livestock production. We examine the main two different aspects of genetic engineering: cloning and transgenesis. After commenting what has been expected from both techniques in livestock production in the last 25 years, the practical difficulties for implementing cloning and transgenesis are examined. Apart from technical difficulties, problems derived from the detection of genetically superior animals and evaluation of the clones and the transgenic animals make these techniques less interesting than they appear to be. Most of the observed variability of the economically interesting traits is not genetic, genetic evaluation needs a large number of animals and cloning success will represent a serious loss of genetic variability and the loss of the flexibility needed for markets in constant evolution. There is a risk in transgenic animals of production of new intermediate biochemical products that may be toxic, allergenic or carcinogenic. The benefits produced by transgenic animals hitherto hardly justify this risk. The expectations that genetic engineering produced 25 years ago should be re-examined, considering the risks and the high investment required.     

精原干细胞介导法制备转基因羊的研究进展

精原干细胞介导法制备转基因动物是用试验导入的方法将外源基因移入动物细胞并整合到其基因组中,伴随着精原干细胞分化成精子,通过受精最终使外源基因得以表达。近年来,随着对精原干细胞研究的不断深入,人们发现其在建立转基因动物方面具有巨大的应用潜力和优势。作者从精原干细胞的生物学特性及携带外源基因的原理等方面阐述了其应用于转基因动物制备的理论机制,同时介绍了精原干细胞介导法在制备转基因动物方面,特别是转基因羊生产中的应用现状。     

Ethical reflections on xenotransplantation]

The success of transplantation and the increasing demand for donor organs have led to an increasingly acute shortage of human organs and tissue for transplantation. In this situation xenotransplantation (cross-species transplantation from animals to humans) is discussed as a possible alternative to allotransplantation. Therefore the animal plays an important role in the discussions on xenotransplantation. Currently, a lot of research is being done on xenotransplantation. Cross-species transplantation involves many biological and medical problems and risks which may affect not only the individual patient but also the wider community and population. These problems give rise to important ethical questions about xenotransplantation. Apart from them other ethical issues are involved, concerning the human being as well as the animal. Animals are raised, genetically modified (transgenic animals) and kept under SPF-conditions before they can be used as organ sources. At the moment they are used as models in xenotransplantation research. My aim is to discuss some of the major ethical, medical and biological problems of xenotransplantation.