共查询到20条相似文献,搜索用时 15 毫秒
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Systems for multigene delivery in mammalian cells, particularly in the context of genome engineering, have gained a lot of attention in biomolecular research and medicine. Initially these methods were based on RNA polymerase II promoters and were used for the production of protein complexes and for applications in cell biology such as reprogramming of somatic cells to stem cells. Emerging technologies such as CRISPR/Cas9-based genome engineering, which enable any alteration at the genomic level of an organism, require additional elements including U6-driven expression cassettes for RNA expression and homology constructs for designed genome modifications. For these applications, systems with high DNA capacity, flexibility and transfer rates are needed. In this article, we briefly give an update on some of recent strategies that facilitate multigene assembly and delivery into mammalian cells. Also, we review applications in various fields of biology that rely on multigene delivery systems. 相似文献
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Moser S Schlatter S Fux C Rimann M Bailey JE Fussenegger M 《Biotechnology progress》2000,16(5):724-735
We present an update on the pTRIDENT multicistronic mammalian expression vectors and their implications in various metabolic engineering and therapeutic applications. The pTRIDENT vector family has been expanded by construction of a new set of pTRIDENT-based vectors containing constitutive promoters of human origin (ubiquitin C and EF-1alpha promoters) and selectable markers (zeocin resistance) and expressing different reporter genes (secreted alkaline phosphatase (SEAP) and the secreted single-chain urokinase-type plasminogen activator (low-M(r) u-PA)). In addition, we have constructed pTRIDENT derivatives with novel streptogramin-repressible and streptogramin-inducible promoters for simultaneous and adjustable expression of three different transgenes. Streptogramin-inducible and tetracycline-repressible pTRIDENT derivatives were used to simultaneously control expression of three fluorescent proteins in mammalian cells: the enhanced cyan fluorescent protein (CFP), the recently isolated red fluorescent protein (RFP, also designated dsRed), and the enhanced yellow fluorescent protein (YFP). Owing to their modular structure, the pTRIDENT vector family represents a construction kit for the design of novel multicistronic expression constructs. 相似文献
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代谢工程利用重组DNA技术、合成生物学、基因组编辑来改变生物体的细胞网络,包括代谢、基因调控和信号网络等。它可以实现加强包括化学品、燃料、化学原料药和其他生物技术产品等代谢物生产的目标,提升生物制造能力与效率。为了梳理和凝练代谢工程30年来的发展状况,《生物工程学报》特组织出版专刊,从代谢工程总体发展、共性技术以及以什么宿主和做什么产品等4个方面展现该领域的发展动态和趋势,并为代谢工程领域的进一步发展提出建设性的意见与展望。 相似文献
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Darrin Kuystermans Britta Krampe Halina Swiderek Mohamed Al-Rubeai 《Cytotechnology》2007,53(1-3):3-22
Significant strides have been made in mammalian cell based biopharmaceutical process and cell line development over the past
years. With several established mammalian host cell lines and expression systems, optimization of selection systems to reduce
development times and improvement of glycosylation patterns are only some of the advances being made to improve cell culture
processes. In this article, the advances pertaining to cell line development and cell engineering strategies are discussed.
An overview of the cell engineering strategies to enhance cellular characteristics by genetic manipulation are illustrated,
focusing on the use of genomics and proteomics tools and their application in such endeavors. Included in this review are
some of the early studies using the ‘omic’ technique to understand cellular mechanisms of product synthesis and secretion,
apoptosis, cell proliferation and the influence of the physicochemical environment. The article highlights the significance
of integrating genomics and proteomics data with the vast amounts of bioprocess data for improved analysis of the biological
pathways involved. Further improvements of the techniques and methodologies used are needed but ultimately, the new cell engineering
strategies should provide great insight into the regulatory networks within the cell in a bioprocess environment and how to
manipulate them to increase overall productivity. 相似文献
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Weber W Marty RR Keller B Rimann M Kramer BP Fussenegger M 《Biotechnology and bioengineering》2002,80(6):691-705
The novel macrolide-inducible and -repressible mammalian gene regulation systems (E.REX) have been cloned into a variety of sophisticated expression configurations including (1) multi-purpose expression vectors, (2) pTRIDENT-based artificial operons, (3) dual-regulated expression strategies for independent control of two different transgenes, (4) autoregulated vectors for one-step installation of adjustable multigene expression, and (5) oncoretroviral and lentiviral plasmids for transduction of macrolide-, streptogramin- and tetracycline-dependent transactivators and production of cell lines supporting independent control of three different transgenes. This vector portfolio represents a construction kit-like toolbox for efficient installation of adjustable gene expression responsive to clinically licensed antibiotics and enables the design of multiregulated multigene metabolic engineering strategies required for biopharmaceutical manufacturing, gene therapy, and tissue engineering. 相似文献
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微生物代谢工程和合成生物学是当今微生物技术领域研究的热点,微生物的生长速度快、容易进行大规模培养;遗传背景清楚、遗传操作简便可靠等性质使其在与人类生活相关的多个领域中起到重要的作用。微生物细胞工厂是指人工设计的能够进行物质生产的微生物代谢体系。许多微生物细胞工厂的构建由于引入多个基因或整条代谢途径,而可能导致代谢失衡、部分代谢中间产物积累等问题,需要使用一定的调控策略加以控制。以下对涉及多个基因作用的微生物细胞工厂中所使用的调控策略,分为若干层次进行了总结和探讨,并对今后多基因控制策略的发展方向进行了预测与展望。 相似文献
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Capitalizing on a proven multicistronic expression vector platform we have designed novel pTRIDENT vectors which (1). enable coordinated expression of three desired transgenes, (2). are size-optimized, (3). take advantage of small highly efficient internal ribosome entry sites of the GTX or Rbm3 type, (4). harbor various sites specific for homing endonucleases facilitating promoter/multicistronic expression unit/polyadenylation site swapping as well as (5). straightforward integration into human HIV-l-based lentiviral expression vectors tailored to contain compatible homing endonucleases. Multicistronic expression profiles of novel pTRIDENT vectors engineered for different tricistronic expression configurations encoding human low-molecular-weight urokinase-type plasminogen activator (u-PA(LMW)) or Bacillus stearothermophilus-derived alpha-amylase (SAMY), human vascular endothelial growth factor (hVEGF), and human placental secreted alkaline phosphatase (SEAP) have been quantified in Chinese hamster ovary cells (CHO-K1), mouse fibroblasts (NIH/3T3), and/or human fibrosarcoma (HT-1080) cells. In addition, a pTRIDENT-derived SAMY-VEGF-SEAP expression cassette transferred into a compatible lentiviral expression vector enabled simultaneous high-level transgene expression following transduction of transgenic lentiviral particles into primary human chondrocytes. 相似文献
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Ralph Bock 《Plant molecular biology》2013,83(1-2):21-31
The engineering of metabolic pathways in plants often requires the concerted expression of more than one gene. While with traditional transgenic approaches, the expression of multiple transgenes has been challenging, recent progress has greatly expanded our repertoire of powerful techniques making this possible. New technological options include large-scale co-transformation of the nuclear genome, also referred to as combinatorial transformation, and transformation of the chloroplast genome with synthetic operon constructs. This review describes the state of the art in multigene genetic engineering of plants. It focuses on the methods currently available for the introduction of multiple transgenes into plants and the molecular mechanisms underlying successful transgene expression. Selected examples of metabolic pathway engineering are used to illustrate the attractions and limitations of each method and to highlight key factors that influence the experimenter’s choice of the best strategy for multigene engineering. 相似文献
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In genomes of higher eukaryotes, adjacent genes often show coordinated regulation of their expression. Compartmentalization
of multiple neighboring genes into a shared chromatin environment can facilitate this coordinated expression. New mapping
techniques have begun to reveal that such multigene chromatin domains are a common feature of fly and mammalian genomes. Many
different types of chromatin domains have been identified based on the genomic binding patterns of various proteins and histone
modifications. In addition, maps of genome–nuclear lamina associations and of looping interactions between loci provide the
first systematic views of the three-dimensional folding of interphase chromosomes. These genome-wide datasets uncover new
architectural principles of eukaryotic genomes and indicate that multigene chromatin domains are prevalent and important regulatory
units. 相似文献
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Engineering of mammalian production cell lines to improve titer and quality of biopharmaceuticals is a top priority of the biopharmaceutical manufacturing industry providing protein therapeutics to patients worldwide. While many engineering strategies have been successful in the past decade they were often based on the over‐expression of a single transgene and therefore limited to addressing a single bottleneck in the cell's production capacity. We provide evidence that ectopic expression of the global metabolic sensor and processing protein mammalian target of rapamycin (mTOR), simultaneously improves key bioprocess‐relevant characteristics of Chinese hamster ovary (CHO) cell‐derived production cell lines such as cell growth (increased cell size and protein content), proliferation (increased cell‐cycle progression), viability (decreased apoptosis), robustness (decreased sensitivity to sub‐optimal growth factor and oxygen supplies) and specific productivity of secreted human glycoproteins. Cultivation of mTOR‐transgenic CHO‐derived cell lines engineered for secretion of a therapeutic IgG resulted in antibody titers of up to 50 pg/cell/day, which represents a four‐fold increase compared to the parental production cell line. mTOR‐based engineering of mammalian production cell lines may therefore have a promising future in biopharmaceutical manufacturing of human therapeutic proteins. Biotechnol. Bioeng. 2011; 108:853–866. © 2010 Wiley Periodicals, Inc. 相似文献
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