Transgenic farm animals - A critical analysis

The notion of directly introducing new genes or otherwise manipulating the genotype of an animal is conceptually straightforward and appealing from the standpoints of both speed and precision with which phenotypic changes can be made. Thus, it is little wonder that the imagination of many animal scientists has been captivated by the success others have achieved in introducing foreign genes into mice. Transgenic mice not only exhibit unique phenotypes, but they also pass those traits on to their progeny. However, before transgenic farm animals become a common component of the livestock industry, a number of formidable obstacles must be overcome. In this review we attempt to identify the critical issues that should be considered by both those currently working in the field and those scientists considering the feasibility of initiating a transgenic livestock project. The inefficiency of producing transgenic animals has been well documented. This does not constrain investigators using laboratory animal models, but it has a major impact on applying transgenic technology to farm animals. The molecular mechanisms of transgene integration have not been elucidated, and as a consequence it is difficult to design strategies to improve the efficiency of the process. In addition to the problems associated with integration of new genes, there are inefficiencies associated with collecting and culturing fertilized eggs as well as embryo transfer in farm animals. Transgenic farm animal studies are major logistical undertakings. Even in the face of these practical hindrances, some may be pressured by administrators to embrace this new technology. As powerful as the transgenic animal model system is, currently there are limits to the kinds of agricultural questions that can be addressed. Some uses are so appealing, however, that several commercial organizations have explored this technology. Within the next decade or two, it is likely that many of the technical hurdles will be overcome. Combining new techniques with a better understanding of the genetic control of physiological systems will make it possible to improve the characteristics of farm animals in highly imaginative ways.     

动物转基因新技术研究进展

动物转基因技术是21世纪发展最为迅速的生物高新技术之一, 它是指通过基因工程技术将外源基因整合到受体动物基因组中, 从而使其得以表达和遗传的生物技术。动物转基因的关键限制因素是转基因效率和基因表达的精确调控。目前有多种转基因技术, 每一种技术各有其优缺点, 仍然需要进一步研究。随着研究的深入, 转基因技术必将在探讨基因功能、动物遗传改良、生物反应器、动物疾病模型、器官移植等领域有广阔的应用前景。文章综述了近年发展的提高转基因效率的生殖干细胞法、提高转基因精确性的基因打靶法、RNA干扰(RNAi)介导的基因沉默技术和诱导多能干细胞(iPS)转基因技术。新的转基因技术为转基因动物的研究提供了更好的平台, 可以加快促进人类医药卫生、畜牧生产等领域的发展。     

The rumen: a unique source of enzymes for enhancing livestock production

Increasing competition in the livestock industry has forced producers to cut costs by adopting new technologies aimed at increasing production efficiency. One particularly promising technology is feeding enzymes as supplements for animal diets. Supplementation of diets for non-ruminants (e.g., swine and poultry) with fibrolytic enzymes, such as cellulases, xylanases and beta-glucanases, increases the feed conversion efficiency and growth rate of the animals. Enzymatic hydrolysis of plant cell wall polymers (e.g., cellulose, xylan, beta-glucans) releases glucose and xylose and eliminates the antinutritional effects of beta-glucans and arabinoxylans. Enzyme supplementation of diets for ruminants has also been shown to improve growth performance, even though the rumen itself represents the most potent fibrolytic fermentation system known. Implementation of this technology in the livestock industry has been limited largely because of the cost of development and production of enzymes. Over the last decade, however, developments in recombinant DNA technology have increased the efficiency of existing microbial production systems and facilitated exploitation of alternative sources of industrial enzymes. The ruminal ecosystem is among the novel enzyme sources currently being explored. Understanding the role of enzymes in feed digestion through characterization of the enzymology and genetics involved in digestion of feedstuffs by ruminants will provide insight required to improve the products currently available to producers. Characterization of genes encoding a variety of hydrolytic enzymes, such as cellulases, xylanases, beta-glucanases, amylases, pectinases, proteases, phytases and tannases, will foster the development of more efficacious enzyme supplements and enzyme expression systems for enhancing nutrient utilization by domestic animals. Characteristics of the original source organism need no longer restrict the production of a useful enzyme. Recent reports of transgenic plants expressing fibrolytic or phytase activity and of transgenic mice able to produce endoglucanase in the pancreas speak to the feasibility of improving feed digestion through genetic modification of the feedstuffs and the animals.     

Developing efficient strategies for the generation of transgenic cattle which produce biopharmaceuticals in milk

At the close of the millennium, a revolution in the treatment of disease is taking shape due to the emergence of new therapies based on human recombinant proteins. The ever-growing demand for such pharmaceutical proteins is an important driving force for the development of safe and large-scale production platforms. Since the efficacy of a human protein is generally dependent on both its amino acid composition as well as various post-translational modifications, many recombinant human proteins can only be obtained in a biologically active conformation when produced in mammalian cells. Hence, mammalian cell culture systems are often used for expression. However, this approach is generally known for limited production capacity and high costs. In contrast, the production of (human) recombinant proteins in milk of transgenic farm animals, particularly cattle, presents a safe alternative without the constraint of limited protein output. Moreover, compared to cell culture, production in milk is very cost-effective. Although transgenic farm animal technology was still in its infancy a decade ago, today it is on the verge of fulfilling its potential of providing therapeutic proteins that can not be produced otherwise in sufficient quantities or at affordable cost. Since 1989, we have been at the forefront of this development, as illustrated by the birth of Herman, the first transgenic bull. In this communication, we will present an overview of approaches we have taken over the years to generate transgenic founder animals and production herds. Our initial strategies were based on microinjection; at the time the only viable option to generate transgenic cattle. Recently, we have adopted a more powerful approach founded on the application of nuclear transfer. As we will illustrate, this strategy presents a breakthrough in the overall efficiency of generating transgenic animals, product consistency, and time of product development.     

The contribution of farm animals to human health

Farm animals and their products have a longstanding and successful history of providing significant contributions to human nutrition, clothing, facilitation of labour, research, development and medicine and have thus been essential in improving life expectancy and human health. With the advent of transgenic technologies the potential of farm animals for improving human health is growing and many areas remain to be explored. Recent breakthroughs in reproductive technologies, such as somatic cloning and in vitro embryo production, and their merger with molecular genetic tools, will further advance progress in this field. Here, we have summarized the contribution of farm animals to human health, covering the production of antimicrobial peptides, dietary supplements or functional foods, animals used as disease models and the contribution of animals to solving urgent environmental problems and challenges in medicine such as the shortage of human cells, tissues and organs and therapeutic proteins. Some of these areas have already reached the level of preclinical testing or commercial application, others will be further advanced only when the genomes of the animals concerned have been sequenced and annotated. Provided the necessary precautions are being taken, the transmission of pathogens from animals to humans can be avoided to provide adequate security. Overall, the promising perspectives of farm animals and their products warrant further research and development in this field.     

提高转基因表达水平的策略

随着转基因相关技术的发展,转基因动物技术在许多方面得到了成功应用.但外源基因在体内的表达仍然难以预测,特别是大动物的转基因,由于制备效率低下,因而难以筛选出足够的高表达的阳性动物数.基因表达调控研究对提高外源基因在动物体内的表达水平提供了一些新手段,本就避免转基因的位置效应、控制外源基因在动物宿主基因组中的整合、提高转基因的表达效率、构建转基因载体和使用外源基因需要注意的问题等进行综述.     

动物乳腺生物反应器的现状和趋势

利用转基因家畜的乳腺生产人类重组蛋白,可以高效获得安全、足量的药用蛋白。本文针对乳腺生物反应器的成功研制,从目的基因的选择、载体构建、转基因技术等方面探讨了动物乳腺生物反应器的研究现状。分析了提高转基因效率和外源蛋白表达水平的技术途径,提出了降低总体成本的战略措施。特别探讨了利用Cre-loxP系统发展“体细胞打靶体细胞核移植技术体系”,高效生产乳腺生物反应器动物的可能性。     

转基因动物克隆技术的应用及生物反应器的建立

利用转基因克隆技术实现外源基因的导入宿主染色体基因组内稳定整合,并能遗传给后代,已在基因表达与调控的理论研究、人类遗传病动物模型的建立、药用蛋白的生产、抗病育种、人类移植用的器官的研究等方面得到广泛应用。转基因动物的研究与应用也已经成为21世纪生命科学领域最活跃、最具有实际应用价值的方向之一,尤其是作为生物反应器和医学上为人类提供所用器官方面,其经济价值和社会效益将是不可估量。在查阅大量近年来国内外相关资料的基础上,本文以转基因动物克隆为中心,对转基因动物克隆所采用显微注射技术、核移植技术、基因打靶与真核BAC表达载体制备等主要研究技术,以及转基因动物克隆在异种器官移植、构建生物反应器等方面的应用进行了综合性论述与分析,同时阐述了各种转基因技术的优点与缺点,以其为转基因动物克隆研究提供理论基础与技术支撑。     

Current status of regulating biotechnology-derived animals in Canada: animal health and food safety considerations

Development of an effective regulatory system for genetically engineered animals and their products has been the subject of increasing discussion among researchers, industry and policy developers, as well as the public. Since transgenesis and cloning are relatively new scientific techniques, transgenic animals are 'novel' organisms for which there is limited information. The issues associated with the regulation of transgenic animals pertain to environmental impact, human food safety, animal health and welfare, trade and ethics. It is a challenge for the developers to prove the safety of the products of biotechnology-derived animals and also for regulators to regulate this increasingly powerful technology with limited background information. In principle, an effective regulatory sieve should permit safe products while forming a formidable barrier for those posing an unacceptable risk. Regulatory initiatives for biotechnology-derived animals and their products should be able to ensure high standards for human and animal health, a sound scientific basis for evaluation; transparency and public involvement, and maintenance of genetic diversity. This review proposes a regulatory regime that is based on scientific risk based assessment and approval of products or by-products of biotechnology-derived animals and its application in context to Canadian regulations.     

YAC transgenesis in farm animals: Rescue of albinism in rabbits

The generation of transgenic mice with mammalian genes cloned in yeast artificial chromosomes (YACs) has generated great interest in the field of gene transfer into livestock. Many of the problems associated with standard transgenesis—such as lack of crucial regulator elements and position effects related to the integration site, which lead to variation in expression levels irrespective of the dose of the transgene—have been practically overcome. The large size of YAC-derived gene constructs (in excess of 1 Mb) facilitates the presence and transfer of all elements required for the faithful regulation of a gene. With the experiments discussed in this report, we have addressed the possibility of applying the obvious advantages of YAC transgenesis to farm animals. We have generated transgenic rabbits carrying a 250 kb YAC covering the mouse tyrosinase gene by pronuclear microinjection, and thus rescued the albino phenotype of the transgenic individuals. To date, this is the first demonstration of a successful transfer of large genetic units into the germ line of farm animals. This development might improve the occurrence of transgene expression at physiological levels and specific sites in livestock. YAC transgenesis therefore will be applied in genetic engineering, for example, in the production of pharmacologically interesting proteins encoded by large gene units and generating transgenic donors for xenotransplantation. © 1996 Wiley-Liss, Inc.     

通过诱导剂作用于启动子的条件性基因表达(英文)

通过遗传工程技术获得的转基因动植物对分析某些生化过程和发育途径极为有用。通过化学诱导剂作用于启动子的条件性基因表达是分子生物学和生物技术应用研究中的强有力的手段。建立于目标基因激活和失活基础之上的几个化学分子诱导基因表达系统已有报道。将来自于原核生物、昆虫和其它动物的调节因子应用于新的物种有利于促进转基因技术的应用和有关基因的时空表达研究。本文综述了有关的基因表达调节系统 ,启动子激活的基因表达系统 ,启动子失活的基因表达系统 ,以及可诱导的基因过度表达和反义抑制系统     

Developments in transgenic technology: applications for medicine

Recent advances in the efficiency of transgenic technology have important implications for medicine. The production of therapeutic proteins from animal bioreactors is well established and the first products are close to market. The genetic modification of pigs to improve their suitability as organ donors for xenotransplantation has been initiated, but many challenges remain. The use of transgenesis, in combination with the method of RNA interference to knock down gene expression, has been proposed as a method for making animals resistant to viral diseases, which could reduce the likelihood of transmission to humans. Here, the latest developments in transgenic technology and their applications relevant to medicine and human health will be discussed.     

Targeting transgene expression in research,agricultural, and environmental applications: Promoters used in plant transformation

Summary Plant genetic engineering has contributed substantially to the understanding of gene regulation and plant development, in the generation of transgenic organisms for widespread usage in agriculture, and has increased the potential uses of crops for industrial and pharmaceutical purposes. As the application of geneticallly engineered plants has widened, so has the need to develop methods to fine-tune control of transgene expression. The availability of a broad spectrum of promoters that differ in their ability to regulate the temporal and spatial expression patterns of the transgene can dramatically increase the successful application of transgenic technology. Indeed, a variety of promoters in necessary at all levels of genetic engineering in plants, from basic research discoveries, concepts and question to development of economically viable crops and plant commodities, to addressing legitimate concerns raised about the safety and containment of transgenic plants in the environment. This review covers the characterization and usage of a broad range of promoters employed in plant genetic engineering, including the widespread use of plant promoters with viral and plant origin that drive constitutive expression. Also covered are selected tissue-specific promoters from fruit, seed and grain, tubers, flowers, pistils, anther and pollen, roots and root nodules, and leaves and green tissue. Topics also include organellar promoters, and those found in specific cell types, as well as the development and evaluation of inducible (endogenous and exogenous origin) and synthetic plant promoter systems. Discussions on the relevance and potential pitfalls within specific applications are included.     

转基因动物研究新进展

我们曾对转基因动物的制作方法、转基因动物研究的应用、转基因的表达特征及提高转基因表达的策略等作过专题综述＾「１」。而这之后在转基因动物的研究方面又获得了许多新进展,其中转线粒体动物的问世拓宽了转基因动物的研究内容。在制作转基因动物的方法上,１９９８－１９９９年世界上先后报道了三种新方法：即由Ｓｃｈｎｉｅｋ等报道的体细胞核移植技术实现转基因,由Ａｎｔｈｏｎｙ Ｗ．Ｓ．Ｃ等报道的通过用逆转录病毒载体感     

Genetic transformation technology: Status and problems

Summary Transfer of genes from heterologous species provides the means of selectively introducing new traits into crop plants and expanding the gene pool beyond what has been available to traditional breeding systems. With the recent advances in genetic engineering of plants, it is now feasible to introduce into crop plants, genes that have previously been inaccessible to the conventional plant breeder, or which did not exist in the crop of interest. This holds a tremendous potential for the genetic enhancement of important food crops. However, the availability of efficient transformation methods to introduce foreign DNA can be a substantial barrier to the application of recombinant DNA methods in some crop plants. Despite significant advances over the past decades, development of efficient transformation methods can take many years of painstaking research. The major components for the development of transgenic plants include the development of reliable tissue culture regeneration systems, preparation of gene constructs and efficient transformation techniques for the introduction of genes into the crop plants, recovery and multiplication of transgenic plants, molecular and genetic characterization of transgenic plants for stable and efficient gene expression, transfer of genes to elite cultivars by conventional breeding methods if required, and the evaluation of transgenic plants for their effectiveness in alleviating the biotic and abiotic stresses without being an environmental biohazard. Amongst these, protocols for the introduction of genes, including the efficient regeneration of shoots in tissue cultures, and transformation methods can be major bottlenecks to the application of genetic transformation technology. Some of the key constraints in transformation procedures and possible solutions for safe development and deployment of transgenic plants for crop improvement are discussed.     

Size Matters: Use of YACs,BACs and PACs in Transgenic Animals

In 1993, several groups, working independently, reported the successful generation of transgenic mice with yeast artificial chromosomes (YACs) using standard techniques. The transfer of these large fragments of cloned genomic DNA correlated with optimal expression levels of the transgenes, irrespective of their location in the host genome. Thereafter, other groups confirmed the advantages of YAC transgenesis and position-independent and copy number-dependent transgene expression were demonstrated in most cases. The transfer of YACs to the germ line of mice has become popular in many transgenic facilities to guarantee faithful expression of transgenes. This technique was rapidly exported to livestock and soon transgenic rabbits, pigs and other mammals were produced with YACs. Transgenic animals were also produced with bacterial or P1-derived artificial chromosomes (BACs/PACs) with similar success. The use of YACs, BACs and PACs in transgenesis has allowed the discovery of new genes by complementation of mutations, the identification of key regulatory sequences within genomic loci that are crucial for the proper expression of genes and the design of improved animal models of human genetic diseases. Transgenesis with artificial chromosomes has proven useful in a variety of biological, medical and biotechnological applications and is considered a major breakthrough in the generation of transgenic animals. In this report, we will review the recent history of YAC/BAC/PAC-transgenic animals indicating their benefits and the potential problems associated with them. In this new era of genomics, the generation and analysis of transgenic animals carrying artificial chromosome-type transgenes will be fundamental to functionally identify and understand the role of new genes, included within large pieces of genomes, by direct complementation of mutations or by observation of their phenotypic consequences.     

The molecular manipulation of milk composition

The introduction of cloned genes into the mouse germ line is now routine. Although more difficult technically, gene transfer has been accomplished in farm animals and offers the potential for genetic improvement. In this regard, we have been investigating the use of transgenic animals as production vehicles for high value proteins in milk. We have shown that DNA sequences derived from the gene encoding sheep beta-lactoglobulin mediate efficient and specific expression in the mammary gland. A fusion gene comprising beta-lactoglobulin sequences and those encoding antihemophilic human factor IX has been constructed. This construct has been introduced into sheep; it is expressed in the mammary gland, and the corresponding protein is secreted into milk.     

Science and technology of farm animal cloning: state of the art

Details of the first mammal born after nuclear transfer cloning were published by Steen Malte Willadsen in 1986. In spite of its enormous scientific significance, this discovery failed to trigger much public concern, possibly because the donor cells were derived from pre-implantation stage embryos. The major breakthrough in terms of public recognition has happened when Ian Wilmut et al. [Wilmut, I., Schnieke, A.E., McWhir, J., Kind, A.J., Campbell, K.H., 1997. Viable offspring derived from fetal és adult mammalian cells. Nature 385, 810-813] described the successful application of almost exactly the same method, but using the nuclei of somatic cells from an adult mammal, to create Dolly the sheep. It has become theoretically possible to produce an unlimited number of genetic replicates from an adult animal or a post-implantation foetus. Since 1997 a number of different species including pigs, goats, horses, cats, etc. have been cloned with the somatic cell nuclear transfer technique. Although the technology still has relatively low success rates and there seems to be substantial problems with the welfare of some of the cloned animals, cloning is used both within basic research and the biomedical sector. The next step seems to be to implement cloning in the agricultural production system and several animals have been developed in this direction. This article reviews the current state of the art of farm animal cloning from a scientific and technological perspective, describes the animal welfare problems and critically assess different applications of farm animal cloning. The scope is confined to animal biotechnologies in which the use of cell nuclear transfer is an essential part and extends to both biomedical and agricultural applications of farm animal cloning. These applications include the production of genetically identical animals for research purposes, and also the creation of genetically modified animals. In the agricultural sector, cloning can be used as a tool within farm animal breeding. We do not intend to give an exhaustive review of the all the literature available; instead we pinpoint issues and events pivotal to the development of current farm animal cloning practices and their possible applications.     

Transgenic animals and nutrition research

Transgenic animals are useful tools for the study of biological functions of proteins and secondary gene products synthesized by the action of protein catalysts. Research in nutrition and allied fields is benefiting from their use as models to contrast normal and altered metabolism. Although food, nutritional products, and ingredients from transgenic animals have not yet reached consumers, the technologies for their production are maturing and yielding exciting results in experimental and farm animals. Regulatory governmental bodies are already issuing guidelines and legislation in anticipation of the advent of these products and ingredients. This review summarizes available technology for the production of transgenic animals, discusses their scientific and commercial potential, and examines ancillary issues relevant to the field of nutrition.