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1.
Houdebine LM 《Comparative immunology, microbiology and infectious diseases》2009,32(2):107-121
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. 相似文献
2.
Wheeler MB 《Journal of animal science》2003,81(Z3):32-37
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. 相似文献
3.
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. 相似文献
4.
《畜牧与生物技术杂志(英文版)》2015,(4)
Handmade cloning(HMC) is the most awaited,simple and micromanipulator-free version of somatic cell nuclear transfer(SCNT).The requirement of expensive micromanipulators and skilled expertise is eliminated in this technique,proving it as a major revolution in the field of embryology.During the past years,many modifications have been incorporated in this technique to boost its efficiency.This alternative approach to micromanipulator based traditional cloning(TC) works wonder in generating comparable or even higher birth rates in addition to declining costs drastically and enabling cryopreservation.This technique is not only applicable to intraspecies nuclear transfer but also to interspecies nuclear transfer(iSCNT) thus permitting conservation of endangered species.It also offers unique possibilities for automation of SCNT which aims at production of transgenic animals that can cure certain human diseases by producing therapeutics hence,providing a healthier future for the wellbeing of humans.The present review aims at highlighting certain aspects of HMC including recent advancements in procedure and factors involved in elevating its efficiency besides covering the potentials and pitfalls of this technique. 相似文献
5.
Bacci ML 《Veterinary research communications》2007,31(Z1):9-14
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. 相似文献
6.
乳腺生物反应器的研究和产业化进展 总被引：3，自引：1，他引：2
随着转基因技术的发展,利用乳腺生物反应器生产珍贵药用蛋白已成为一种极具潜力的生物技术,它为商业化前景广阔的药用蛋白提供了高产量、低成本的生产方式,开创了基因工程制药的新途径。目前,应用乳腺生物反应器已表达百余种药用蛋白,其中近30种进入临床试验不同阶段,用于治疗抗凝血酶缺乏症的抗凝血酶Ⅲ（ATrynR）已上市,乳腺生物反应器生产重组蛋白显示出广阔的产业化前景。作者概述了乳腺生物反应器的原理、优点、国内外研究进展和产业化进程,讨论了其目前存在问题和应对策略。 相似文献
7.
Somatic cell nuclear transfer (SCNT, 'cloning') holds great potential for agricultural applications, generation of medical model animals, transgenic farm animals or by 'therapeutic cloning' for generating human embryonic stem cells for the treatment of human diseases. However, the low survival rate of SCNT-derived pregnancies represents a serious limitation of the current technology. In order to overcome this hurdle, a deeper understanding of the epigenetic reprogramming of the somatic cell nuclei and its effect on the pregnancy is needed. Here we review the literature on nuclear reprogramming by SCNT, including studies of gene expression, DNA methylation, chromatin remodelling, genomic imprinting and X chromosome inactivation. Reprogramming of genes expressed in the inner cell mass, from which the body of the foetus is formed, seems to be highly efficient. Defects in the extra-embryonic tissues are probably the major cause of the low success rate of reproductive cloning. Methods to partially overcome such problems exist, yet more future research is needed to find practical and efficient methods to remedy this problem. Improvement of the survival of foetuses is a central issue for the future of agricultural SCNT not only for its economic viability, but also because in lack of improvements in animal welfare current regulations can block the use of the method in the EU and several other countries. 相似文献
8.
Liebenberg J Pretorius A Faber FE Collins NE Allsopp BA van Kleef M 《Veterinary immunology and immunopathology》2012,145(1-2):340-349
Ehrlichia ruminantium is an obligate intracellular bacterial pathogen which causes heartwater, a serious tick-borne disease of ruminants throughout sub-Saharan Africa. The development of promising recombinant vaccines has been reported previously, but none has been as effective as immunisation with live organisms. In this study we have used reverse vaccinology to identify proteins that elicit an in vitro cellular immune response similar to that induced by intact E. ruminantium. The experimental strategy involved four successive steps: (i) in silico selection of the most likely vaccine candidate genes from the annotated genome; (ii) cloning and expression of the selected genes; (iii) in vitro screening of the expressed proteins for their ability to induce interferon-gamma (IFN-γ) production in E. ruminantium-immune lymphocytes; and (iv) further examination of the cytokine response profiles of those lymphocytes which tested positive for IFN-γ induction. Based on their overall cytokine induction profiles the recombinant proteins were divided into four distinct groups. Eleven recombinant proteins induced a cytokine profile that was similar to the recall immune response induced by immune peripheral blood mononuclear cells (PBMC) stimulated with intact E. ruminantium. This response comprised the upregulation of cytokines associated with adaptive cellular immune responses as well as innate immunity. A successful vaccine may therefore need to contain a combination of recombinant proteins which induce both immune pathways to ensure protection against heartwater. 相似文献
9.
Honscha W 《DTW. Deutsche tier?rztliche Wochenschrift》1999,106(10):425-432
A modern pharmacotherapy without the use of proteins as drugs is not more practicable. In the clinic blood coagulation factors (factor VIII and IX), growth hormones (human growth hormone), enzymes (1-antitrysin, insulin), and cytokines are currently used. At the beginning, the proteins were isolated from biological sources or expressed in vitro by the use of E. coli or eukaryotic cell lines. At the moment efforts were undertaken to express these proteins in the milk of transgenic animals. This review article describes the methods for the generation of transgenic animals, the benefits and drawbacks of this new technique in comparison to established methods for the production of proteins as pharmaceuticals. At the end of the review possible improvements of the method are described. 相似文献
10.
L-M Houdebine 《Reproduction in domestic animals》2005,40(4):269-281
Contents Transgenic animals are more widely used for various purposes. Applications of animal transgenesis may be divided into three major categories: (i) to obtain information on gene function and regulation as well as on human diseases, (ii) to obtain high value products (recombinant pharmaceutical proteins and xeno-organs for humans) to be used for human therapy, and (iii) to improve animal products for human consumption. All these applications are directly or not related to human health. Animal transgenesis started in 1980. Important improvement of the methods has been made and are still being achieved to reduce cost as well as killing of animals and to improve the relevance of the models. This includes gene transfer and design of reliable vectors for transgene expression. This review describes the state of the art of animal transgenesis from a technical point of view. It also reports some of the applications in the medical field based on the use of transgenic animal models. The advance in the generation of pigs to be used as the source of organs for patients and in the preparation of pharmaceutical proteins from milk and other possible biological fluids from transgenic animals is described. The projects in course aiming at improving animal production by transgenesis are also depicted. Some the specific biosafety and bioethical problems raised by the different applications of transgenesis, including consumption of transgenic animal products are discussed. 相似文献
11.
Pedro MOREIRA Serafín PéREZ-CEREZALES Ricardo LAGUNA Raúl FERNáNDEZ-GONZALEZ Belén Pintado SANJUANBENITO Alfonso GUTIéRREZ-ADáN 《The Journal of reproduction and development》2016,62(1):37-42
The production of transgenic animals is an important tool for experimental and applied biology. Over the
years, many approaches for the production of transgenic animals have been tried, including pronuclear
microinjection, sperm-mediated gene transfer, transfection of male germ cells, somatic cell nuclear transfer
and the use of lentiviral vectors. In the present study, we developed a new transgene delivery approach, and
we report for the first time the production of transgenic animals by co-injection of DNA and round spermatid
nuclei into non-fertilized mouse oocytes (ROSI). The transgene used was a construct containing the human CMV
immediate early promoter and the enhanced GFP gene. With this procedure, 12% of the live offspring we obtained
carried the transgene. This efficiency of transgenic production by ROSI was similar to the efficiency by
pronuclear injection or intracytoplasmic injection of male gamete nuclei (ICSI). However, ICSI required fewer
embryos to produce the same number of transgenic animals. The expression of Egfp mRNA and
fluorescence of EGFP were found in the majority of the organs examined in 4 transgenic lines generated by
ROSI. Tissue morphology and transgene expression were not distinguishable between transgenic animals produced
by ROSI or pronuclear injection. Furthermore, our results are of particular interest because they indicate
that the transgene incorporation mediated by intracytoplasmic injection of male gamete nuclei is not an
exclusive property of mature sperm cell nuclei with compact chromatin but it can be accomplished with immature
sperm cell nuclei with decondensed chromatin as well. The present study also provides alternative procedures
for transgene delivery into embryos or reconstituted oocytes. 相似文献
12.
Cloning and kinetics of expression of Brucella abortus heat shock proteins by baculovirus recombinants 总被引：1，自引：0，他引：1
In an effort to develop genetically engineered Brucella abortus (BA) vaccines, the genes encoding heat shock proteins (HSPs) GroEL, GroES, and HtrA were cloned and expressed in the BAC-TO-BAC Baculovirus System, and the kinetics of protein expression were analyzed using various insect cell lines in suspension cultures, different cell densities in suspension cultures, multiplicities of infection and recombinant virus replication times. Trichoplusia ni cells expressed only BA HtrA, but Spodoptera frugiperda (Sf9) cells expressed all three recombinant proteins. The best GroEL expression was achieved by infecting 2x10(6) Sf9 cells/ml with an MOI 10 of recombinant virus and harvesting the cells after 96h of virus replication. GroES and HtrA were best expressed when infecting 2x10(6) Sf9 cells/ml with an MOI 1 of recombinant viruses and harvesting the cells after 120h of virus replications. Under these conditions BA recombinant HSPs were expressed as follows: GroEL at 16% of the total cellular proteins (105microg/ml concentration); GroES 2% (15.25microg/ml); and HtrA 8% (84.48microg/ml). This is the first report of cloning and expression of BA genes in the baculovirus system. 相似文献
13.
利用动物生物反应器生产重组蛋白是一种具有应用前景的生物技术。鸡输卵管生物反应器是理想的动物生物反应器之一,其优点在于表达的外源蛋白能够分泌到蛋清中,可避免蛋白提取过程中对鸡本身造成伤害,同时蛋清成分简单,便于后期的纯化。目前利用慢病毒结合原始生殖细胞(PGCs)制备转基因鸡被认为是最可行的方法,但因外源基因随机整合且生殖系传递效率较低,使转基因鸡研究受到技术上的限制。而2013年问世的CRISPR/Cas9基因敲入(CRISPR/Cas9 knock-in)技术能够使外源基因精准定向插入基因组特异性位点,这对生产输卵管特异性转基因鸡具有重大意义。文章综述了鸡输卵管反应器的研究进展、CRISPR/Cas9 knock-in技术在输卵管特异性表达转基因鸡研究和鸡育种领域的应用现状,并指出了目前存在的问题和相应的解决办法。 相似文献
14.
Petersen B Carnwath JW Niemann H 《Comparative immunology, microbiology and infectious diseases》2009,32(2):91-105
The shortage of donated human organs for transplantation continues to be a life threatening problem for patients suffering from complete organ failure. Although this gap is increasing due to the demographic changes in aging Western populations, it is generally accepted that international trading in human organ is not an ethical solution. Alternatives to the use of human organs for transplantation must be developed and these alternatives include stem cell therapy, artificial organs and organs from other species, i.e. xenografts. For practical reasons but most importantly because of its physiological similarity with humans, the pig is generally accepted as the species of choice for xenotransplantation. Nevertheless, before porcine organs can be used in human xenotransplantation, it is necessary to make a series of precise genetic modifications to the porcine genome, including the addition of genes for factors which suppress the rejection of transplanted porcine tissues and the inactivation or removal of undesirable genes which can only be accomplished at this time by targeted recombination and somatic nuclear transfer. This review will give an insight into the advances in transgenic manipulation and cloning in pigs--in the context of porcine-to-human xenotransplantation. 相似文献
15.
Van Reenen CG Meuwissen TH Hopster H Oldenbroek K Kruip TH Blokhuis HJ 《Journal of animal science》2001,79(7):1763-1779
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. 相似文献
16.
Hirabayashi M 《The Journal of reproduction and development》2008,54(2):95-99
Transgenic rats have been used as model animals for human diseases and organ transplantation and as animal bioreactors for protein production. In general, transgenic rats are produced by pronuclear microinjection of exogenous DNA. Improvement of post-injection survival has been achieved by micro-vibration of the injection pipette. The promoter region, structural gene, chain length and strand ends of the exogenous DNA are not involved in the production efficiency of transgenic rats. Exogenous DNA prepared at 5 microg/ml seemed to be better integrated than lower and higher concentrations. Intracytoplasmic sperm injection (ICSI) has been successfully achieved in rats using a piezo-driven injection pipette. The ICSI technique has not only been applied to rescue infertile male strains but also to produce transgenic rats. The optimal DNA concentration for the ICSI-tg method (0.1 to 0.5 microg/ml) is lower than that for the conventional pronuclear microinjection. Production efficiency was improved when the membrane structure of the sperm head was partially disrupted by detergent or ultrasonic treatment before exposure to the exogenous DNA solution. For successful production of transgenic rats with a modified endogenous gene, establishment of embryonic stem cell lines or alternatively male germline stem cell lines and technical development of somatic cell nuclear transfer are still necessary for this species. 相似文献
17.
The recombinant DNA technology or DNA cloning permits the isolation, amplification, and precise manipulation of specific DNA fragments. This is generally accomplished by linking or recombining the desired DNA fragment with a DNA molecule, termed the vector, which is capable of directing the replication of itself in a suitable host cell and any DNA segment covalently attached to it. Using this and associated technologies, it is possible to produce large amounts of specific proteins and to modify cell types by introducing the genes for proteins that are otherwise absent. Moreover, it is now possible to construct variants of naturally-occurring proteins with improved biological or physical properties. 相似文献
18.
Modern animal breeding programs are largely based on biotechnological procedures, including AI and embryo transfer technology. Recent breakthroughs in reproductive technologies, such as somatic cell nuclear transfer and in vitro embryo production, and their combination with the emerging molecular genetic tools, will further advance progress and provide new opportunities for livestock breeding. This is urgently needed in light of the global challenges such as the ever-increasing human population, the limited resources of arable land, and the urgent environmental problems associated with farm animal production. Here, we focus on genomic breeding strategies and transgenic approaches for making farm animals more feed efficient. Based on studies in the mouse and rat model, we have identified a panel of genes that are critically involved in the regulation of feed uptake and that could contribute toward future breeding of farm animals with reduced environmental impact. We anticipate that genetically modified animals will play a significant role in shaping the future of feed-efficient and thus sustainable animal production, but will develop more slowly than the biomedical applications because of the complexity of the regulation of feed intake and metabolism. 相似文献
19.
20.
Sox2是多能干细胞的主要标记之一,有研究发现高表达Sox2基因的神经干细胞作为供体细胞进行核移植时具有较高的重编程能力。本研究旨在通过对绵羊骨髓间充质干细胞(bonemarrowmesenchymal stem cells,BMSC) Sox2基因进行外源性增强表达,以期提高其重编程能力,从而改善动物体细胞克隆效率。试验提取绵羊胎儿生殖腺组织RNA,以其为模板克隆Sox2基因cDNA序列,装入真核表达载体pEGFP-N1,构建出pEGFP-N1-Sox2表达载体。经脂质体转染将重组质粒转染入绵羊BMSC,经G418与荧光标记双筛选后挑选单克隆并扩增培养。测序鉴定表明,克隆得到绵羊Sox2基因CDS区全长,重组质粒构建成功;荧光检测表明,成功建立表达Sox2基因的绵羊BMSC系。本研究得到了高表达Sox2基因的绵羊BMSC系,为提高体细胞克隆过程中的重编程效率提供了新思路。 相似文献