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。     

Lactation performance of transgenic goats expressing recombinant human butyryl-cholinesterase in the milk

The production of recombinant proteins in the milk of transgenic animals has attracted significant interest in the last decade, as a valuable alternative for the production of recombinant proteins that cannot be or are inefficiently produced using conventional systems based on microorganisms or animal cells. Several recombinant proteins of pharmaceutical and biomedical interest have been successfully expressed in high quantities (g/l) in the milk of transgenic animals. However, this productivity may be associated with a compromised mammary physiology resulting, among other things, from the extraordinary demand placed on the mammary secretory cells. In this study we evaluated the lactation performance of a herd of 50 transgenic goats expressing recombinant human butyryl-cholinesterase (rBChE) in the milk. Our findings indicate that high expression levels of rBChE (range 1–5 g/l) are produced in these animals at the expense of an impaired lactation performance. The key features characterizing these transgenic performances were the decreased milk production, the reduced milk fat content which was associated with an apparent disruption in the lipid secretory mechanism at the mammary epithelium level, and a highly increased presence of leukocytes in milk which is not associated with mammary infection. Despite of having a compromised lactation performance, the amount of rBChE produced per transgenic goat represents several orders of magnitude more than the amount of rBChE present in the blood of hundreds of human donors, the only other available source of rBChE for pharmaceutical and biodefense applications. As a result, this development constitutes another successful example in the application of transgenic animal technology.     

Downstream processing of recombinant proteins from transgenic feedstock

The search for inexpensive production systems capable of producing large quantities of recombinant protein has resulted in the development of new technology platforms based on transgenic plants and animals. Over the past decade, these transgenic systems have been used to produce several products and potential therapeutic proteins. Improvements continue to be made, not only in how the proteins are expressed but also in how the end products are obtained. As improvements in expression are realized, cost-saving measures will increasingly focus on downstream processing.     

Transgenic animal bioreactors

The production of recombinant proteins is one of the major successes of biotechnology. Animal cells are required to synthesize proteins with the appropriate post-translational modifications. Transgenic animals are being used for this purpose. Milk, egg white, blood, urine, seminal plasma and silk worm cocoon from transgenic animals are candidates to be the source of recombinant proteins at an industrial scale. Although the first recombinant protein produced by transgenic animals is expected to be in the market in 2000, a certain number of technical problems remain to be solved before the various systems are optimized. Although the generation of transgenic farm animals has become recently easier mainly with the technique of animal cloning using transfected somatic cells as nuclear donor, this point remains a limitation as far as cost is concerned. Numerous experiments carried out for the last 15 years have shown that the expression of the transgene is predictable only to a limited extent. This is clearly due to the fact that the expression vectors are not constructed in an appropriate manner. This undoubtedly comes from the fact that all the signals contained in genes have not yet been identified. Gene constructions thus result sometime in poorly functional expression vectors. One possibility consists in using long genomic DNA fragments contained in YAC or BAC vectors. The other relies on the identification of the major important elements required to obtain a satisfactory transgene expression. These elements include essentially gene insulators, chromatin openers, matrix attached regions, enhancers and introns. A certain number of proteins having complex structures (formed by several subunits, being glycosylated, cleaved, carboxylated...) have been obtained at levels sufficient for an industrial exploitation. In other cases, the mammary cellular machinery seems insufficient to promote all the post-translational modifications. The addition of genes coding for enzymes involved in protein maturation has been envisaged and successfully performed in one case. Furin gene expressed specifically in the mammary gland proved to able to cleave native human protein C with good efficiency. In a certain number of cases, the recombinant proteins produced in milk have deleterious effects on the mammary gland function or in the animals themselves. This comes independently from ectopic expression of the transgenes and from the transfer of the recombinant proteins from milk to blood. One possibility to eliminate or reduce these side-effects may be to use systems inducible by an exogenous molecule such as tetracycline allowing the transgene to be expressed only during lactation and strictly in the mammary gland. The purification of recombinant proteins from milk is generally not particularly difficult. This may not be the case, however, when the endogenous proteins such as serum albumin or antibodies are abundantly present in milk. This problem may be still more crucial if proteins are produced in blood. Among the biological contaminants potentially present in the recombinant proteins prepared from transgenic animals, prions are certainly those raising the major concern. The selection of animals chosen to generate transgenics on one hand and the elimination of the potentially contaminated animals, thanks to recently defined quite sensitive tests may reduce the risk to an extremely low level. The available techniques to produce pharmaceutical proteins in milk can be used as well to optimize milk composition of farm animals, to add nutriceuticals in milk and potentially to reduce or even eliminate some mammary infectious diseases. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Reestablishment of a transgenic rabbit line by artificial insemination using cryopreserved semen

The production of recombinant proteins in the milk of transgenic animals is an alternative to traditional cell culture methodology. Transgenic rabbits can serve in the small-scale production of recombinant proteins, underscoring the need to maintain valuable transgenic lines. In this study, the authors used cryopreserved transgenic rabbit semen to artificially inseminate does, demonstrating the utility of this method for the reestablishment of a transgenic rabbit herd.     

Antibody molecular farming in plants and plant cells

`Molecular Farming' is a novel approach to the production of pharmaceuticals, where valuable recombinant proteins can be produced in transgenic organisms on an agricultural scale. Plants have been traditionally used as a source of medicines, but the use of transgenic plants in molecular farming represents a novel source of molecular medicines that include plasma proteins, enzymes, growth factors, vaccines and recombinant antibodies. Until recently, the wide use of these molecular medicines was limited because of the difficulty in producing these proteins outside animals or animal cell cultures. The application of molecular biology and plant biotechnology in the 1990s showed that many molecular medicines could be synthesised in plants. The goal of this Molecular Farming technology is to produce pharmaceuticals that are safer, easier to produce and less expensive than those produced in animals or microbial cultures. Here, we examine the production of recombinant antibodies by Molecular Farming.     

Effect of genetic background on glycosylation heterogeneity in human antithrombin produced in the mammary gland of transgenic goats

Glycosylation is involved in the correct folding, targeting, bioactivity and clearance of therapeutic glycoproteins. With the development of transgenic animals as expression systems it is important to understand the impact of different genetic backgrounds and lactations on glycosylation. We have evaluated the glycosylation of recombinant antithrombin produced in several transgenic goat lines, from cloned animals and from different types of lactation including induced lactations. Our results show glycosylation patterns from the protein expressed in animals, derived from the same founder goat, are mostly comparable. Furthermore, the protein expressed in two cloned goats had highly consistent oligosaccharide profiles and similar carbohydrate composition. However, there were significantly different oligosaccharide profiles from the proteins derived from different founder goats. Artificial induction of lactation did not have significant effects on overall carbohydrate structures when compared to natural lactation. The only major difference was that recombinant antithrombin from induced lactations contained a slightly higher ratio of N-acetylneuraminic acid to N-glycolylneuraminic acid and less amount of oligosaccharides containing N-glycolylneuraminic acid. The oligosaccharides from all animals were a mixture of high mannose-, hybrid- and complex-type oligosaccharides. Sialic acid was present as alpha-2,6-linkage and no alpha-1,3-linked galactose was observed. These results indicate that transgenic animals with closely related genetic backgrounds express recombinant protein with comparable glycosylation.     

Antibody manufacture in transgenic animals and comparisons with other systems

Various forms of recombinant monoclonal antibodies are being used increasingly, mainly for therapeutic purposes. The isolation and engineering of the corresponding genes is becoming less of a bottleneck in the process; however, the production of recombinant antibodies is itself a limiting factor and a shortage is expected in the coming years. Milk from transgenic animals appears to be one of the most attractive sources of recombinant antibodies. None of the production systems presently implemented (CHO cells, insect cells infected by baculovirus, or transgenic animals and plants) has yet been optimized. This review describes the advantages of using milk for antibody production in comparison with the other systems.     

动物乳腺生物反应器研究进展

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。本在简要介绍乳腺生物反应器的基本原理及优越性的基础上,对其目的基因、表达载体和转基因技术在国内外的研究现状加以综述,着重探讨了体细胞核移植方法生产乳腺生物反应器的优越性及面临的技术问题。     

Biopharmaceutical production in transgenic livestock.

The production of recombinant human proteins in the milk of transgenic dairy animals offers a safe, renewable source of commercially important proteins that cannot be produced as efficiently in adequate quantities by other methods. A decade of success in expressing a variety of proteins in livestock has brought three human recombinant proteins to human clinical trials. Recent progress has drawn on molecular biology and reproductive physiology to improve the efficiency of producing and reproducing useful transgenic founder animals, and to improve the expression of heterologous proteins in their milk.     

Posttranslational modification of therapeutic proteins in plants

Plants have emerged as an alternative to current systems for the production of therapeutic proteins. The advantages of plants for the low-cost and large-scale production of safe and biologically active mammalian proteins have been documented recently. A major advantage of transgenic plants over production systems that are based on yeast or Escherichia coli is their ability to perform most of the posttranslational modifications (PTMs) that are required for the bioactivity and pharmacokinetics of recombinant therapeutic proteins. Furthermore, recent advances in the control of PTMs in transgenic plants have made it possible for plants to perform, at least to some extent, human-like modifications of recombinant proteins. Hence, plants have become a suitable alternative to animal cell factories for the production of therapeutic proteins.     

Production of recombinant proteins by microbes and higher organisms

Large proteins are usually expressed in a eukaryotic system while smaller ones are expressed in prokaryotic systems. For proteins that require glycosylation, mammalian cells, fungi or the baculovirus system is chosen. The least expensive, easiest and quickest expression of proteins can be carried out in Escherichia coli. However, this bacterium cannot express very large proteins. Also, for S–S rich proteins, and proteins that require post-translational modifications, E. coli is not the system of choice. The two most utilized yeasts are Saccharomyces cerevisiae and Pichia pastoris. Yeasts can produce high yields of proteins at low cost, proteins larger than 50 kD can be produced, signal sequences can be removed, and glycosylation can be carried out. The baculoviral system can carry out more complex post-translational modifications of proteins. The most popular system for producing recombinant mammalian glycosylated proteins is that of mammalian cells. Genetically modified animals secrete recombinant proteins in their milk, blood or urine. Similarly, transgenic plants such as Arabidopsis thaliana and others can generate many recombinant proteins.     

Production of therapeutic proteins in algae,analysis of expression of seven human proteins in the chloroplast of Chlamydomonas reinhardtii

Recombinant proteins are widely used today in many industries, including the biopharmaceutical industry, and can be expressed in bacteria, yeasts, mammalian and insect cell cultures, or in transgenic plants and animals. In addition, transgenic algae have also been shown to support recombinant protein expression, both from the nuclear and chloroplast genomes. However, to date, there are only a few reports on recombinant proteins expressed in the algal chloroplast. It is unclear whether this is because of few attempts or of limitations of the system that preclude expression of many proteins. Thus, we sought to assess the versatility of transgenic algae as a recombinant protein production platform. To do this, we tested whether the algal chloroplast could support the expression of a diverse set of current or potential human therapeutic proteins. Of the seven proteins chosen, >50% expressed at levels sufficient for commercial production. Three expressed at 2%–3% of total soluble protein, while a forth protein accumulated to similar levels when translationally fused to a well‐expressed serum amyloid protein. All of the algal chloroplast‐expressed proteins are soluble and showed biological activity comparable to that of the same proteins expressed using traditional production platforms. Thus, the success rate, expression levels, and bioactivity achieved demonstrate the utility of Chlamydomonas reinhardtii as a robust platform for human therapeutic protein production.     

Expression of eukaryotic recombinant proteins and deriving them from the milk of transgenic animals

Expression in milk of transgenic farm animals is one of the most effective ways to produce eukaryotic recombinant proteins. In this review, we discuss the problems of the appropriateness of selecting this expression system for the production of proteins and the development characteristics of appropriate production processes.     

The transgenic animal platform for biopharmaceutical production

The recombinant production of therapeutic proteins for human diseases is currently the largest source of innovation in the pharmaceutical industry. The market growth has been the driving force on efforts for the development of new therapeutic proteins, in which transgenesis emerges as key component. The use of the transgenic animal platform offers attractive possibilities, residing on the low production costs allied to high productivity and quality of the recombinant proteins. Although many strategies have evolved over the past decades for the generation of transgenic founders, transgenesis in livestock animals generally faces some challenges, mainly due to random transgene integration and control over transgene copy number. But new developments in gene editing with CRISPR/Cas system promises to revolutionize the field for its simplicity and high efficiency. In addition, for the final approval of any given recombinant protein for animal or human use, the production and characterization of bioreactor founders and expression patterns and functionality of the proteins are technical part of the process, which also requires regulatory and administrative decisions, with a large emphasis on biosafety. The approval of two mammary gland-derived recombinant proteins for commercial and clinical use has boosted the interest for more efficient, safer and economic ways to generate transgenic founders to meet the increasing demand for biomedical proteins worldwide.     

Efficiency of human lactoferrin transgenic donor cell preparation for SCNT

The combination of somatic cell nuclear transfer (SCNT) and transgenic technology leads to the production of transgenic cloned animals, wherein the preparation of competent transgenic donor cells is the pivotal upstream step. The purpose of this study was to establish an efficient procedure to prepare human lactoferrin (hLTF) transgenic donor cells for SCNT. Thus, two cell culture systems were employed: caprine mammary epithelial cells (for evaluation of the hTLF transgenic expression in vitro), and fetal-derived fibroblast cells (for identification of competent transgenic donor cells). Induced by hormonal signals, recombinant hLTF was detected in the supernatant of transfected mammary epithelial cells by Western blot. Reliable hLTF transgenic fibroblast cell clones were identified by screening with multiple PCR amplification, EGFP fluorescence, and chromosomal counting (32.5+/-2.3%). This study may provide an effective upstream system to prepare SCNT donor cells for the production of human recombinant pharmaceuticals from the milk of transgenic animals.     

Commercial exploitation of transgenics

A series of examples of the application of recombinant nucelic acid technology to the production of transgenic organisms is presented. The review considers the distinct advantages of producing certain recombinant biomolecules in appropriate eucaryotic systems rather than in the traditional procaryotic ones. Gene therapy in humans and the production of transgenic animals and plants are discussed. Concerns about the inadvertant construction of new pathogens and ethical considerations about interfereing with poorly understood ecosystems are addressed.     

Milk composition studies in transgenic goats expressing recombinant human butyrylcholinesterase in the mammary gland

The use of the mammary gland of transgenic goats as a bioreactor is a well established platform for the efficient production of recombinant proteins, especially for molecules that cannot be adequately produced in traditional systems using genetically engineered microorganisms and cells. However, the extraordinary demand placed on the secretory epithelium by the expression of large amounts of the recombinant protein, may result in a compromised mammary physiology. In this study, milk composition was compared between control and transgenic goats expressing high levels (1-5 g/l) of recombinant human butyrylcholinesterase in the milk. Casein concentration, as evaluated by acid precipitation, was significantly reduced in the transgenic compared with the control goats throughout lactation (P < 0.01). Milk fatty acid composition for transgenic goats, as determined by gas chromatography, was found to have significantly fewer short chain fatty acids (P < 0.01) and more saturated fatty acids (P < 0.05) compared to controls, suggesting an overall metabolic stress and/or decreased expression of key enzymes (e.g. fatty acid synthase, stearoyl-CoA desaturase). The concentration of Na(+), K(+), assessed by atomic absorption spectrophotometry, and serum albumin, determined by bromocresol green dye and scanning densitometry, were similar in transgenic and control goats during the first several weeks of lactation. However, as lactation progressed, a significant increase in Na and serum albumin concentrations and a decrease in K(+) concentration were found in the milk of transgenic goats, while control animals remained unchanged (P < 0.01). These findings suggest that: (a) high expression of recombinant proteins may be associated with a slow-down in other synthetic activities at the mammary epithelium, as evidenced by a reduced casein expression and a decreased de-novo synthesis of fatty acids; (b) the development of permeable tight junctions may be the main mechanism involved in the premature cessation of milk secretion observed in these transgenic goats.     

人促血小板生成素在转基因小鼠乳腺中定位表达的研究(英文)

通过转基因动物乳腺生物反应器大规模生产药用蛋白质已成为现代生物技术新的生长点之一。为研制表达人促血小板生成素的哺乳动物生物反应器的转基因小鼠模型,本论文以小鼠乳清酸蛋白 (mWAP) 基因5挾说骺厍团-s1-酪蛋白基因3挾说骺厍魑鹘谠菇擞糜诒泶锶舜傺“迳伤氐娜橄僮橹匾煨员泶镌靥錺WAPTPO(Fig.1)。通过常规显微注射的方法把mWAP启动子指导的hTPO表达载体导入小鼠受精卵,获得出生小鼠16只。经PCR检测,有6只为转基因阳性(Fig.2)。G0代小鼠中转基因整合率为37.5% (6/16),用ELISA方法在G0代转基因雌鼠的乳汁中检测了促血小板生成素的表达,表达量在0.8 mg/mL以上(Table 1)。这些结果表明我们已建立了乳腺表达hTPO 的转基因小鼠模型,为以后大型家畜乳腺生物反应器的研制提供了科学依据。