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1.
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. 相似文献
2.
Lactation performance of transgenic goats expressing recombinant human butyryl-cholinesterase in the milk 总被引:1,自引:0,他引:1
Baldassarre H Hockley DK Doré M Brochu E Hakier B Zhao X Bordignon V 《Transgenic research》2008,17(1):73-84
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. 相似文献
3.
High level expression of a functional human/mouse chimeric anti-CD20 monoclonal antibody in milk of transgenic mice 总被引:2,自引:1,他引:1
Rituximab, a chimeric anti-CD20 monoclonal antibody, is one of the most successful biomedicines and has been used to treat at least 370,000 patients with indolent, aggressive non-Hodgkin's lymphoma and other malignant diseases. However, the global demand for rituximab and other therapeutic monoclonal antibodies is exponentially increasing and barely able to be met by current manufacturing capacities of mammalian cell culture. The mammary gland bioreactor has been regarded as an ideal substitute for mammalian cell culture to mass-produce recombinant monoclonal antibodies at the lowest possible cost. Here, we show a feasible model to produce recombinant anti-CD20 antibodies in the mammary glands of transgenic animals. Six lines of transgenic mice were generated by co-microinjection of the two expression cassettes that can specially express the chimeric light and heavy chain of anti-CD20 mAbs in the milk of transgenic animals. The recombinant antibodies were detected in the milk of transgenic mice with the highest expression level up to 17 microg/mul and could specifically bind the CD20 surface antigens on human B-lymphoma cells. 相似文献
4.
Antibody molecular farming in plants and plant cells 总被引:1,自引:0,他引:1
`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. 相似文献
5.
'Molecular farming' is the production of recombinant proteins in plants. It is intended to harness the power of agriculture to cultivate and harvest transgenic plants producing recombinant therapeutics. Molecular farming has the potential to provide virtually unlimited quantities of recombinant antibodies for use as diagnostic and therapeutic tools in both health care and the life sciences. Importantly, recombinant antibody expression can be used to modify the inherent properties of plants, for example by using expressed antipathogen antibodies to increase disease resistance. Plant transformation is technically straightforward for model plant species and some cereals, and the functional expression of recombinant proteins can be rapidly analyzed using transient expression systems in intact or virally infected plants. Protein production can then be increased using plant suspension cell production in fermenters, or by the propagation of stably transformed plant lines in the field. Transgenic plants can be exploited to produce organs rich in a recombinant protein for its long-term storage. This demonstrates the promise of using transgenic plants as bioreactors for the 'molecular farming' of recombinant therapeutics, blood substitutes and diagnostics, such as recombinant antibodies. 相似文献
6.
Generation and production of engineered antibodies 总被引:7,自引:0,他引:7
Various forms of recombinant monoclonal antibodies are being used increasingly, mainly for therapeutic purposes. This review
specifically focuses on what is now called antibody engineering, and discusses the generation of chimeric, humanized, and
fully human recombinant antibodies, immunoglobulin fragments, and artificial antigen-binding molecules. Since the production
of recombinant antibodies is a limiting factor in their availability, and a shortage is expected in the future, different
expression systems for recombinant antibodies and transgenic organisms as bioreactors are also discussed, along with their
advantages and drawbacks. 相似文献
7.
《MABS-AUSTIN》2013,5(6):1138-1150
In response to the successful use of monoclonal antibodies (mAbs) in the treatment of various diseases, systems for expressing recombinant mAbs using transgenic animals or plants have been widely developed. The silkworm (Bombyx mori) is a highly domesticated insect that has recently been used for the production of recombinant proteins. Because of their cost-effective breeding and relatively easy production scale-up, transgenic silkworms show great promise as a novel production system for mAbs. In this study, we established a transgenic silkworm stably expressing a human-mouse chimeric anti-CD20 mAb having the same amino acid sequence as rituximab, and compared its characteristics with rituximab produced by Chinese hamster ovary (CHO) cells (MabThera®). The anti-CD20 mAb produced in the transgenic silkworm showed a similar antigen-binding property, but stronger antibody-dependent cell-mediated cytotoxicity (ADCC) and weaker complement-dependent cytotoxicity (CDC) compared to MabThera. Post-translational modification analysis was performed by peptide mapping using liquid chromatography/mass spectrometry. There was a significant difference in the N-glycosylation profile between the CHO? and the silkworm-derived mAbs, but not in other post-translational modifications including oxidation and deamidation. The mass spectra of the N-glycosylated peptide revealed that the observed biological properties were attributable to the characteristic N-glycan structures of the anti-CD20 mAbs produced in the transgenic silkworms, i.e., the lack of the core-fucose and galactose at the non-reducing terminal. These results suggest that the transgenic silkworm may be a promising expression system for the tumor-targeting mAbs with higher ADCC activity. 相似文献
8.
Minoru Tada Ken-Ichiro Tatematsu Akiko Ishii-Watabe Akira Harazono Daisuke Takakura Noritaka Hashii Hideki Sezutsu Nana Kawasaki 《MABS-AUSTIN》2015,7(6):1138-1150
In response to the successful use of monoclonal antibodies (mAbs) in the treatment of various diseases, systems for expressing recombinant mAbs using transgenic animals or plants have been widely developed. The silkworm (Bombyx mori) is a highly domesticated insect that has recently been used for the production of recombinant proteins. Because of their cost-effective breeding and relatively easy production scale-up, transgenic silkworms show great promise as a novel production system for mAbs. In this study, we established a transgenic silkworm stably expressing a human-mouse chimeric anti-CD20 mAb having the same amino acid sequence as rituximab, and compared its characteristics with rituximab produced by Chinese hamster ovary (CHO) cells (MabThera®). The anti-CD20 mAb produced in the transgenic silkworm showed a similar antigen-binding property, but stronger antibody-dependent cell-mediated cytotoxicity (ADCC) and weaker complement-dependent cytotoxicity (CDC) compared to MabThera. Post-translational modification analysis was performed by peptide mapping using liquid chromatography/mass spectrometry. There was a significant difference in the N-glycosylation profile between the CHO− and the silkworm-derived mAbs, but not in other post-translational modifications including oxidation and deamidation. The mass spectra of the N-glycosylated peptide revealed that the observed biological properties were attributable to the characteristic N-glycan structures of the anti-CD20 mAbs produced in the transgenic silkworms, i.e., the lack of the core-fucose and galactose at the non-reducing terminal. These results suggest that the transgenic silkworm may be a promising expression system for the tumor-targeting mAbs with higher ADCC activity. 相似文献
9.
Developing efficient strategies for the generation of transgenic cattle which produce biopharmaceuticals in milk 总被引:20,自引:0,他引:20
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. 相似文献
10.
The plant vesicular transport engineering for production of useful recombinant proteins 总被引:4,自引:0,他引:4
Kazuya Yoshida Takeshi Matsui Atsuhiko Shinmyo 《Journal of Molecular Catalysis .B, Enzymatic》2004,28(4-6):167-171
The molecular breeding of plants that have been genetically engineered for improved disease resistance and stress tolerance has been undertaken with the goal of improving food production. More recently, it has been realized that transgenic plants can serve as bioreactors for the production of proteins or compounds with industrial or clinical uses. Several different recombinant enzymes and antibodies have been produced in this manner. To maximize the potential of industrial plants as a production system for proteins, efficient expression systems utilizing promoters that optimize transgene expression, 5′-untranslated region elements for efficient translation, and appropriate post-translational modifications and localization must be developed. This review summarizes successful examples of the production of recombinant enzymes, antibodies, and vaccines using signal peptides that direct vesicular localization in transgenic plants. We further discuss the modulation of recombinant protein localization to the endoplasmic reticulum, vacuolar system, or extracellular compartments by varying the signal peptide. 相似文献
11.
Koles K van Berkel PH Mannesse ML Zoetemelk R Vliegenthart JF Kamerling JP 《Glycobiology》2004,14(11):979-986
The large-scale production of recombinant biopharmaceutical glycoproteins in the milk of transgenic animals is becoming more widespread. However, in comparison with bacterial, plant cell, or cell culture production systems, little is known about the glycosylation machinery of the mammary gland, and hence on the glycosylation of recombinant glycoproteins produced in transgenic animals. Here the influence is presented of several lactation parameters on the N-glycosylation of recombinant C1 inhibitor (rhC1INH), a human serum glycoprotein, expressed in the milk of transgenic rabbits. Enzymatically released N-glycans of series of rhC1INH samples were fluorescently labeled and fractionated by HPLC. The major N-glycan structures on rhC1INH of pooled rabbit milk were similar to those on native human C1 inhibitor and recombinant human C1 inhibitor produced in transgenic mouse milk, with only the degree of sialylation and core fucosylation being lower. Analyses of individual animals furthermore showed slight interindividual differences; a decrease in the extent of sialylation, core fucosylation, and oligomannose-type glycosylation with the progress of lactation; and a positive correlation between expression level and oligomannose-type N-glycan content. However, when large quantities of rhC1INH were isolated for preclinical and clinical studies, highly consistent N-linked glycan profiles and monosaccharide compositions were found. 相似文献
12.
Antibody production by molecular farming in plants 总被引:7,自引:0,他引:7
Fischer R Hoffmann K Schillberg S Emans N 《Journal of biological regulators and homeostatic agents》2000,14(2):83-92
"Molecular farming" is the production of pharmaceutical proteins in transgenic plants and has great potential for the production of therapeutic anti-cancer antibodies and recombinant therapeutic proteins. Plants make fully functional recombinant human or animal antibodies. Cultivating transgenic plants on an agricultural scale will produce almost unlimited supplies of recombinant proteins for uses in medicine. Combinatorial library technology is a key tool for the generation and optimisation of therapeutic antibodies ahead of their expression in plants. Optimised antibody expression can be rapidly verified using transient expression assays in plants before creation of transgenic suspension cells or plant lines. Subcellular targeting signals that increase expression levels and optimise protein stability can be identified and exploited using transient expression to create high expresser plant lines. When high expresser lines have been selected, the final step is the development of efficient purification methods to retrieve functional antibody. Antibody production on an industrial scale is then possible using plant suspension cell culture in fermenters, or by the propagation of stably transformed plant lines in the field. Recombinant proteins can be produced either in whole plants or in seeds and tubers, which can be used for the long-term storage of both the protein and its production system. The review will discuss these developments and how we are moving toward the molecular farming of therapeutic antibodies becoming an economic and clinical reality. 相似文献
13.
Smith TJ 《Biotechnology advances》1994,12(4):679-686
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. 相似文献
14.
动物乳腺生物反应器的现状和趋势 总被引:19,自引:1,他引:19
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。 相似文献
15.
Transgenic animal bioreactors 总被引:24,自引:2,他引:22
Houdebine LM 《Transgenic research》2000,9(4-5):305-320
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. 相似文献
16.
Jingjing Wei Xiangmin Yang Min Zheng Meili Wang Yunping Dai Zhinan Chen Ning Li 《Transgenic research》2011,20(2):321-330
Biologically active recombinant monoclonal antibodies (mAbs) and their derivatives are in demand as therapeutic agents against
a variety of cancers. The antibodies are generally produced by mammalian cell culture, but their production in the milk of
transgenic animals would help meet the increasing demand. The mouse-human chimeric antibody chHAb18 has been proven to inhibit
the invasion and metastasis of human hepatocellular carcinoma (HCC) cells by recognizing the HAb18G/CD147 molecule that is
highly expressed on the surface of HCC tissue. Here, we report that transgenic mice generated by co-microinjection of two
cassettes encoding the heavy and light chain genes of chHAb18 could highly express functional chHAb18 in their mammary glands.
The expression level range of 1.1–7.4 mg ml−1 was independent of transgenic copy number. Immunoassays demonstrated the ability and specificity of chHAb18 to bind purified
antigen (i.e., HAb18G) or HCC cells. Recombinant chHAb18 from transgenic milk exhibited affinity almost equal to chHAb18 derived
from CHO cells, and was 68% of that of the parental murine antibody, HAb18. In light of successful clinical application of
HAb18, the chHAb18 expressed in mammary glands of transgenic mice constitutes an important step towards high-yield and scaled-up
production of this antibody. 相似文献
17.
Transgenic animal bioreactors represent a powerful tool to address the growing need for therapeutic recombinant proteins. The ability of transgenic animals to produce complex, biologically active recombinant proteins in an efficient and economic manner has stimulated a great deal of interest in this area. As a result, genetically modified animals of several species, expressing foreign proteins in various tissues, are currently being developed. However, the generation of transgenic animals is a cumbersome process and remains problematic in the application of this technology. The advantages and disadvantages of different transgenic systems in relation to other bioreactor systems are discussed. 相似文献
18.
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. 相似文献
19.
Phytopharming, the production of protein biologicals in recombinant plant systems, has shown great promise in studies performed over the past 13 years. A secretory antibody purified from transgenic tobacco was tested successfully in humans, and prevented bacterial re-colonization after topical application in the mouth. Rapid production of patient-tailored anti-lymphoma antibodies in recombinant Tobamovirus-infected tobacco may provide effective cancer therapy. Many different candidate vaccines from bacterial and viral sources have been expressed in transgenic plants, and three human clinical trials with oral delivery of transgenic plant tissues have shown exciting results. The use of crop plants with agricultural practice could allow cheap production of valuable proteins, while providing enhanced safety by avoidance of animal viruses or other contaminants. However development of this technology must carefully consider the means to ensure the separation of food and medicinal products when crop plants are used for phytopharming. 相似文献
20.
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. 相似文献