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Gene-Specific and Genome-Wide ChIP Approaches to Study Plant Transcriptional NetworksEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Chromatin immunoprecipitation (ChIP) provides a versatile tool to investigate the in vivo location of DNA-binding proteins on genomic DNA. ChIP approaches are gaining significance in plants, in cases when entire genome sequences are available (e.g., Arabidopsis), for which several high-density oligo arrays have been or are being developed. Nevertheless, plant ChIP and ChIP-chip still present some technical challenges. Here, we describe general methods for ChIP and ChIP-chip, which have been successfully applied to maize and Arabidopsis.
Source: Springer protocols feed by Plant Sciences - January 1, 2009 Category: Biology Source Type: info

Genome-Wide Analysis of RNA–Protein Interactions in PlantsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
RNA–protein interactions profoundly impact organismal development and function through their contributions to the basal gene expression machineries and their regulation of post-transcriptional processes. The repertoire of predicted RNA binding proteins (RBPs) in plants is particularly large, suggesting that the RNA–protein interactome in plants may be more complex and dynamic even than that in metazoa. To dissect RNA–protein interaction networks, it is necessary to identify the RNAs with which each RBP interacts and to determine how those interactions influence RNA fate and downstream processes. Identific...
Source: Springer protocols feed by Plant Sciences - January 1, 2009 Category: Biology Source Type: info

Whole-Genome Microarrays: Applications and Technical IssuesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
DNA microarrays have become a mainstream tool in experimental plant biology. The constant improvements in the technological platforms have enabled the development of the tiling DNA microarrays that cover the whole genome, which in turn catalyzed the wide variety of creative applications of such microarrays in the areas as diverse as global studies of genetic variation, DNA-binding proteins, DNA methylation, and chromatin and transcriptome dynamics. This chapter attempts to summarize such applications as well as discusses some technical and strategic issues that are particular to the use of tiling microarrays.
Source: Springer protocols feed by Plant Sciences - January 1, 2009 Category: Biology Source Type: info

Manipulating Large-Scale Arabidopsis Microarray Expression Data: Identifying Dominant Expression Patterns and Biological Process EnrichmentEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
A series of large-scale Arabidopsis thaliana microarray expression experiments profiling genome-wide expression across different developmental stages, cell types, and environmental conditions have resulted in tremendous amounts of gene expression data. This gene expression is the output of complex transcriptional regulatory networks and provides a starting point for identifying the dominant transcriptional regulatory modules acting within the plant. Highly co-expressed groups of genes are likely to be regulated by similar transcription factors. Therefore, finding these co-expressed groups can reduce the dimensionality of c...
Source: Springer protocols feed by Plant Sciences - January 1, 2009 Category: Biology Source Type: info

Applications of Ultra-high-Throughput SequencingEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The genomics era has enabled scientists to more readily pose truly global questions regarding mutation, evolution, gene and genome structure, function, and regulation. Just as Sanger sequencing ushered in a paradigm shift that enabled the molecular basis of biological questions to be directly addressed, to an even greater degree, ultra-high-throughput DNA sequencing is poised to dramatically change the nature of biological research. New sequencing technologies have opened the door for novel questions to be addressed at the level of the entire genome in the areas of comparative genomics, systems biology, metagenomics, and g...
Source: Springer protocols feed by Plant Sciences - January 1, 2009 Category: Biology Source Type: info

Isolation of Plant Polysomal mRNA by Differential Centrifugation and Ribosome Immunopurification MethodsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Polyribosomes (polysomes) form as multiple ribosomes engage in translation on a single mRNA. This process is regulated for individual mRNAs by both development and the environment. To evaluate the translation state of an mRNA, ribosomal subunits, ribosomes, and polysomes can be isolated from detergent-treated cell extracts by high-speed differential centrifugation. These ribonucleoprotein complexes can be further purified by centrifugation through sucrose density gradients. By fractionation of the gradient the amount of an individual mRNA in a sub-population of polysomes can be quantitatively determined. Here, we describe ...
Source: Springer protocols feed by Plant Sciences - January 1, 2009 Category: Biology Source Type: info

Chromatin Charting: Global Mapping of Epigenetic EffectsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
To tackle the question of how chromatin organization is involved in global regulation of genome-related processes such as transcription, we have recently created a collection of 277 transposon-tagged Arabidopsis lines comprised of a single insert with a common luciferase reporter cassette and a LacO repeat array for visual tracking of the tagged region via fluorescent protein fusion technology. Using this collection of plants, one can begin to map transgene position effects as well as global epigenetic control in response to developmental or externally applied cues. In this chapter, we will outline the approach and methods...
Source: Springer protocols feed by Plant Sciences - January 1, 2009 Category: Biology Source Type: info

Clone-Based Functional GenomicsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Annotated genomes have provided a wealth of information about gene structure and gene catalogs in a wide range of species. Taking advantage of these developments, novel techniques have been implemented to investigate systematically diverse aspects of gene and protein functions underpinning biology processes. Here, we review functional genomics applications that require the mass production of cloned sequence repertoires, including ORFeomes and silencing tag collections. We discuss the techniques employed in large-scale cloning projects and we provide an up-to-date overview of the clone resources available for model plant sp...
Source: Springer protocols feed by Plant Sciences - January 1, 2009 Category: Biology Source Type: info

Discrete Dynamic Modeling with Asynchronous Update, or How to Model Complex Systems in the Absence of Quantitative InformationEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
A major aim of systems biology is the study of the inter-relationships found within and between large biological data sets. Here we describe one systems biology method, in which the tools of network analysis and discrete dynamic (Boolean) modeling are used to develop predictive models of cellular signaling in cases where detailed temporal and kinetic information regarding the propagation of the signal through the system is lacking. This approach is also applicable to data sets derived from some other types of biological systems, such as transcription factor-mediated regulation of gene expression during the control of devel...
Source: Springer protocols feed by Plant Sciences - January 1, 2009 Category: Biology Source Type: info

Quantification of Variation in Expression NetworksEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Gene expression microarrays allow rapid and easy quantification of transcript accumulation for almost transcripts present in a genome. This technology has been utilized for diverse investigations from studying gene regulation in response to genetic or environmental fluctuation to global expression QTL (eQTL) analyses of natural variation. Typical analysis techniques focus on responses of individual genes in isolation of other genes. However, emerging evidence indicates that genes are organized into regulons, i.e., they respond as groups due to individual transcription factors binding multiple promoters, creating what is co...
Source: Springer protocols feed by Plant Sciences - January 1, 2009 Category: Biology Source Type: info

Co-expression Analysis of Metabolic Pathways in PlantsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Co-expression analysis allows experimenters to re-use archived expression microarray data to uncover previously unknown functional relationships between genes. An observation that a group of genes are co-expressed across diverse experimental conditions suggests they may play similar roles in the cell. Several thousand expression microarray experiments performed on samples from Arabidopsis thaliana have entered the public domain and it is now possible to use these data to investigate metabolic networks in plants. This chapter explains how to use a Web-based tool (CressExpress) to investigate co-expression of genes involved ...
Source: Springer protocols feed by Plant Sciences - January 1, 2009 Category: Biology Source Type: info

Integration of Metabolic Reactions and Gene RegulationEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Metabolic reactions and gene regulation are two primary processes of cells. In response to environmental changes cells often adjust the regulatory programs and shift the metabolic states. An integrative investigation and modeling of these two processes would improve our understanding of the cellular systems and may generate substantial impacts in medicine, agriculture, environmental protection, and energy. We review the studies of the various aspects of the crosstalk between metabolic reactions and gene regulation, including models, empirical evidence, and available databases.
Source: Springer protocols feed by Plant Sciences - January 1, 2009 Category: Biology Source Type: info

Applying Word-Based Algorithms: The IMEterEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Important patterns can be found in strings of characters such as nucleotides in a DNA sequence by examining the frequency of occurrence of specific character combinations or words. The abundance of words can reveal the presence of underlying trends governing the order of characters, even if the biological reasons for those trends remain mysterious. As an example of one way in which word frequencies have provided insight, we describe the IMEter, a word-based algorithm for analyzing introns and their effect on gene expression. The IMEter demonstrates that introns located near the beginning of genes are compositionally distin...
Source: Springer protocols feed by Plant Sciences - January 1, 2009 Category: Biology Source Type: info

Challenges and Approaches to Statistical Design and Inference in High-Dimensional InvestigationsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Advances in modern technologies have facilitated high-dimensional experiments (HDEs) that generate tremendous amounts of genomic, proteomic, and other “omic” data. HDEs involving whole-genome sequences and polymorphisms, expression levels of genes, protein abundance measurements, and combinations thereof have become a vanguard for new analytic approaches to the analysis of HDE data. Such situations demand creative approaches to the processes of statistical inference, estimation, prediction, classification, and study design. The novel and challenging biological questions asked from HDE data have resulted in many...
Source: Springer protocols feed by Plant Sciences - January 1, 2009 Category: Biology Source Type: info

Live-Imaging and Image Processing of Shoot Apical Meristems of Arabidopsis thalianaEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The shoot apical meristem (SAM) of higher plants represents a dynamic network of different cell types which exhibit distinct patterns of gene expression and cellular behaviors. The regulation of distinct patterns of gene expression and cellular behaviors is mediated by cell–cell communication networks. Live-imaging of spatiotemporal dynamics of cell–cell communication networks, gene expression patterns, and cellular behaviors is critical to deduce principles that underlie SAM growth and maintenance. In this chapter, we describe live-imaging methods, fluorescent reagents, and image processing protocols that have...
Source: Springer protocols feed by Plant Sciences - January 1, 2009 Category: Biology Source Type: info

Computer Vision as a Tool to Study Plant DevelopmentEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Morphological phenotypes due to mutations frequently provide key information about the biological function of the affected genes. This has long been true of the plant Arabidopsis thaliana, though phenotypes are known for only a minority of this model organism's approximately 25,000 genes. One common explanation for lack of phenotype in a given mutant is that a genetic redundancy masks the effect of the missing gene. Another possibility is that a phenotype escaped detection or manifests itself only in a certain unexamined condition. Addressing this potentially nettlesome alternative requires the development of more sophisti...
Source: Springer protocols feed by Plant Sciences - January 1, 2009 Category: Biology Source Type: info

Metabolomics of Plant VolatilesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Plants communicate with their surrounding ecosystems using a diverse array of volatile metabolites that are indicative of the physiological status of the emitter. A variety of systems have been adapted to capture, analyze, identify, and quantify airborne metabolites released by plants. Metabolomic experiments typically involve four steps: sample collection, preparation, product separation, and data analysis. To date, two different types of headspace sampling, static and dynamic, are widely used for volatile metabolome investigation. For static headspace analysis, solid-phase microextraction (SPME) is used to sample volatil...
Source: Springer protocols feed by Plant Sciences - January 1, 2009 Category: Biology Source Type: info

Chemical Genomics Approaches in Plant BiologyEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Chemical genomics (i.e., genomics-scale chemical genetics) approaches are based on the ability of low-molecular-mass molecules to modify biological processes. Such molecules are used to affect the activity of a protein or a pathway in a manner that is tunable and reversible. A major advantage of this approach compared to classical plant genetics is the fact that chemical genomics can address loss-of-function lethality and redundancy. Bioactive chemicals resulting from forward or reverse chemical screens can be useful in understanding and dissecting complex biological processes due to the essentially limitless variation in ...
Source: Springer protocols feed by Plant Sciences - January 1, 2009 Category: Biology Source Type: info

Comparison of Quantitative Metabolite Imaging Tools and Carbon-13 Techniques for FluxomicsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The recent development of analytic technologies allows fast analysis of metabolism in real time. Fluxomics aims to define the genes involved in regulation of flux through a metabolic or signaling pathway. Flux through a metabolic or signaling pathway is determined by the activity of its individual components; regulation can occur at many levels, including transcriptional, posttranslational, and allosteric levels. Currently two technologies are used to monitor fluxes. The first is pulse labeling of the organism with a tracer such as C13, followed by mass spectrometric analysis of the partitioning of label into different com...
Source: Springer protocols feed by Plant Sciences - January 1, 2009 Category: Biology Source Type: info

Democratization and Integration of Genomic Profiling ToolsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Systems biology is a comprehensive means of creating a complete understanding of how all components of an organism work together to maintain and procreate life. By quantitatively profiling one at a time, the effect of thousands and millions of genetic and environmental perturbations on the cell, systems biologists are attempting to recreate and measure the effect of the many different states that have been explored during the 3 billion years in which life has evolved. A key aspect of this work is the development of innovative new approaches to quantify changes in the transcriptome, proteome, and metabolome. In this chapter...
Source: Springer protocols feed by Plant Sciences - January 1, 2009 Category: Biology Source Type: info

Analysis of miRNA ModificationsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
After transcription, a large number of cellular RNAs employ modifications to increase their diversity and functional potential. Modifications can occur on the base, ribose, or both, and are important steps in the maturation of many RNAs. Our lab recently showed that plant microRNAs (miRNAs) possess a 2′-O-methyl group on the ribose of the 3′ terminal nucleotide, and that this methyl group is added after miRNA/miRNA* formation. One function of this modification is to protect miRNAs from 3′ terminal uridylation by an unknown enzymatic activity. It is possible that uridylation of miRNAs triggers their degrad...
Source: Springer protocols feed by Plant Sciences - January 1, 2009 Category: Biology Source Type: info

MicroRNA Promoter AnalysisEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this chapter, we present a brief overview of current knowledge about the promoters of plant microRNAs (miRNAs), and provide a step-by-step guide for predicting plant miRNA promoter elements using known transcription factor binding motifs. The approach to promoter element prediction is based on a carefully constructed collection of Positional Weight Matrices (PWMs) for known transcription factors (TFs) in Arabidopsis. A key concept of the method is to use scoring thresholds for potential binding sites that are appropriate to each individual transcription factor. While the procedure can be applied to search for Transcript...
Source: Springer protocols feed by Plant Sciences - January 1, 2009 Category: Biology Source Type: info

Computational Methods for Comparative Analysis of Plant Small RNAsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Small RNAs play an important role in plant development, stress responses, and epigenetic regulation, primarily through their role in transcriptional and post-transcriptional silencing of specific target genes and loci. Most if not all plants utilize these small RNA signaling networks. We have developed a deep-sequencing based dataset of plant small RNAs, based on the hypothesis that comparisons among the complex pool of small RNAs from diverse plants will identify novel types of conserved, regulated, or species-specific molecules. A database containing upward of hundreds of millions of plant small RNA sequences is being cr...
Source: Springer protocols feed by Plant Sciences - January 1, 2009 Category: Biology Source Type: info

Biotic Stress-Associated microRNAs: Identification, Detection, Regulation, and Functional AnalysisEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The methods described herein first highlight the strategies that were used to discover a biotic stress-associated miRNA. This involved (1) the selection of transcripts that were more abundant in transgenic plants expressing viral-derived suppressors of RNA silencing and transcripts that were repressed in wild-type seedlings treated with a biotic stress, (2) a 5′ RACE-derived assay to map miRNA target sites, and (3) a bioinformatic analysis to retrieve specific miRNA loci from the Arabidopsis genome. We then describe methods used to monitor (1) the levels of primary miRNA transcripts (pri-miRNAs)/mature miRNAs and (2)...
Source: Springer protocols feed by Plant Sciences - January 1, 2009 Category: Biology Source Type: info

Abiotic Stress-Associated miRNAs: Detection and Functional AnalysisEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
MicroRNAs (miRNAs) are small regulatory noncoding RNAs varying in length between 20 and 24 nucleotides. They play a key role during plant development by negatively regulating gene expression at the posttranscriptional level. Moreover, recent studies reported several miRNAs associated with abiotic stress responses. Small RNA cloning and high-throughput deep sequencing methods provide expression profiles of not only known miRNAs, but also novel miRNAs. In this chapter, we describe the methods used to identify and characterize abiotic stress-associated miRNAs and their target genes.
Source: Springer protocols feed by Plant Sciences - January 1, 2009 Category: Biology Source Type: info

Erwinia amylovora: Modern Methods for Detection and DifferentiationEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We describe a real-time PCR procedure that provides all requirements. This method is based on chromosomal genes rather than on the pEa29 plasmid and so can be used to measure isolates that have been cured of the plasmid. The method has been used very successfully in directly quantify whole E. amylovora cells, in a variety of tissues from the orchard environment.
Source: Springer protocols feed by Plant Sciences - December 1, 2008 Category: Biology Source Type: info

The use of Fluorescent in situ Hybridisation in Plant Fungal Identification and GenotypingEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
FISH is a widely used technique in many laboratories not only for cytogenetic studies, but also in other biological fields. It requires a combination of skills in molecular biology, cytogenetics, immunocytochemistry, microscopy and cellular imaging analysis.
Source: Springer protocols feed by Plant Sciences - December 1, 2008 Category: Biology Source Type: info

Use of Molecular Methods for the Detection of Fungal SporesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Traditional methods for the isolation and identification of fungal spores can be time-consuming and laborious. DNA-based methods for fungal detection can be used to detect the spores of plant-pathogenic fungi. Air borne spores can be collected and identified by PCR allowing identification of the species.
Source: Springer protocols feed by Plant Sciences - December 1, 2008 Category: Biology Source Type: info

Identification of Phytophthora fragariae var. rubi by PCREmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The following chapter describes a PCR method for the identification of the raspberry root rot pathogen Phytophthora fragariae var. rubi. Furthermore, a nested PCR suitable for the detection of the pathogen in infected raspberry roots and validated against the “Duncan bait test” (EPPO Bull 35:87–91, 2005) is explained. Protocols for different DNA extraction methods are given which can be transferred to other fungal pathogens.
Source: Springer protocols feed by Plant Sciences - December 1, 2008 Category: Biology Source Type: info

Detection of Double-Stranded RNA Elements in the Plant Pathogenic Fungus Rhizoctonia solaniEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Many species of fungi have been shown to harbor double-stranded RNA (dsRNA) elements. A single fungal isolate of Rhizoctonia solani may have as many as five different dsRNA elements within them. The presence of specific dsRNA elements influence pathogenicity in host plants.
Source: Springer protocols feed by Plant Sciences - December 1, 2008 Category: Biology Source Type: info

From Neanderthal to Nanobiotech: From Plant Potions to Pharming with Plant FactoriesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Plants were the main source for human drugs until the beginning of the nineteenth century when plantderived pharmaceuticals were partly supplanted by drugs produced by the industrial methods of chemical synthesis. During the last decades of the twentieth century, genetic engineering has offered an alternative to chemical synthesis, using bacteria, yeasts and animal cells as factories for the production of therapeutic proteins. After a temporary decrease in interest, plants are rapidly moving back into human pharmacopoeia, with the recent development of plant-based recombinant protein production systems offering a safe and ...
Source: Springer protocols feed by Plant Sciences - June 1, 2008 Category: Biology Source Type: info

Chloroplast-Derived Vaccine Antigens and Biopharmaceuticals: Protocols for Expression, Purification, or Oral Delivery and Functional EvaluationEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Many vaccine antigens and biopharmaceutical proteins have been expressed at high levels via the chloroplast genome and their functionality has been evaluated using in vitro assays in cell cultures (i.e., macrophage lysis assay, inhibition of vesicular stomatitis virus-induced cytopathicity in baby hamster kidney cells, or inhibition of human HIV infection in TZM-BL cells) as well as protection after challenge with bacterial or viral pathogens or antitumor assays or delay the onset of insulitis in suitable animal models. Production of therapeutic proteins in chloroplasts eliminates the expensive fermentation technology. Mor...
Source: Springer protocols feed by Plant Sciences - June 1, 2008 Category: Biology Source Type: info

Protein Body Induction: A New Tool to Produce and Recover Recombinant Proteins in PlantsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We present a novel approach to produce recombinant proteins in plants based on the ability of γ zein-Zera domain to store recombinant proteins inside PBs. Zera domain fused to several proteins, including a enhanced cyan fluorescent protein (ECFP), calcitonin (Ct) and epidermal growth factor (EGF), were cloned into vectors for transient or stable transformation of tobacco plants. In tobacco leaves, we observed the formation of dense, ER-localized structures containing high concentrations of the respective target proteins. The intact synthetic organelles containing Zera fusions were readily isolated from cellular mater...
Source: Springer protocols feed by Plant Sciences - June 1, 2008 Category: Biology Source Type: info

A Case Study for Plant-Made Pharmaceuticals Comparing Different Plant Expression and Production SystemsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Over the last decade, plant-based production of pharmaceuticals has made remarkable progress as the expression of a diverse set of proteins has been demonstrated in a range of plant crops. Although the commercial exploitation is still pending, today various plant-based expression technologies have reached significant milestones through clinical testing in humans. Each of the protein manufacturing platforms in plants has specific benefits and drawbacks. We have engaged in comparing some of these production systems with respect to their performance: protein yield and quality. Using a specific tester protein (aprotinin), it w...
Source: Springer protocols feed by Plant Sciences - June 1, 2008 Category: Biology Source Type: info

Glycosylation of Antibody Therapeutics: Optimisation for PurposeEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Recombinant antibody therapeutics represent a significant success story in terms of clinical benefit delivered and revenue (profit) generated within the biopharmaceutical industry. Additionally, it is estimated that ̃30% of new drugs likely to be licensed during the next decade will be based on antibody products. High volume production with the maintenance of structural and functional fidelity of these large biological molecules results in high “cost of goods” that can limit their availability to patients, due to the strain it puts on national and private health budgets. The challenge in reducing cost of g...
Source: Springer protocols feed by Plant Sciences - June 1, 2008 Category: Biology Source Type: info

N-Glycosylation of Plant Recombinant PharmaceuticalsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
N-glycosylation is a maturation event necessary for the correct function, efficiency, and stability of a high number of biopharmaceuticals. This chapter presented here proposes various methods to determine whether, how, and where a plant pharmaceutical is N-glycosylated. These methods rely on blot detection with glycan-specific probes, specific deglycosylation of glycoproteins followed by mass spectrometry, N-glycan profile analysis, and glycopeptide identification by LC-MS.
Source: Springer protocols feed by Plant Sciences - June 1, 2008 Category: Biology Source Type: info

Companion Protease Inhibitors to Protect Recombinant Proteins in Transgenic Plant ExtractsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We describe a general approach for the use of recombinant protease inhibitors as stabilizing agents for clinically useful proteins extracted from transgenic plant tissues. A procedure is first described to assess the overall (in)stability of heterologous proteins in transgenic plant crude protein extracts. Step-by-step protocols are then presented for the choice and use of companion protease inhibitors inhibiting the host plant proteases during extraction. This strategy, that reproduces the protein-stabilizing effect of low-molecular-weight protease inhibitors often added to protein extraction media, does not require the e...
Source: Springer protocols feed by Plant Sciences - June 1, 2008 Category: Biology Source Type: info

Strategies for Improving Vaccine Antigens Expression in Transgenic Plants: Fusion to Carrier SequencesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Transgenic plants are gaining increasing attention from the industry as a natural bioreactor for the production of industrial and chemical products. Optimization of transgene expression in plant cells holds the key to maximizing the potential of plants for producing proteins of commercial interest. This chapter is devoted to the description of the methods utilized for the generation of transgenic plants expressing a canine parvovirus vaccine peptide or virus-like particles from a rabbit calicivirus.
Source: Springer protocols feed by Plant Sciences - June 1, 2008 Category: Biology Source Type: info

Immunomodulation of Plant Function by In Vitro Selected Single-Chain Fv IntrabodiesEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this chapter, we discuss and compare the different concepts and examples as well as present the basic protocols for applying intrabody-based approaches in plants for the investigation of cell functions and plant cell–pathogen interactions. The immunomodulation strategy, a molecular technique that allows to interfere with cellular metabolism, signal transduction pathways, or pathogen infectivity, is based on the ectopic expression of genes encoding specific recombinant antibodies. This needs basic prerequisites to be successfully applied as resources and techniques to isolate specific recombinant antibodies with su...
Source: Springer protocols feed by Plant Sciences - June 1, 2008 Category: Biology Source Type: info

On-Chip Detection of Low-Molecular-Weight Recombinant Proteins in Plant Crude Extracts by SELDI-TOF MSEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
This chapter presents a general procedure for the on-chip detection and quantitation of low-molecular-weight recombinant proteins in transgenic plant crude extracts by surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS). A protocol is first described to detect the protein of interest in crude protein extracts of transgenic plant lines, by differential protein mapping against similar extracts from a control, nontransgenic line. A complementary protocol is then presented to generate a standard curve with the SELDI system, allowing the protein to be quantified in different transgenic l...
Source: Springer protocols feed by Plant Sciences - June 1, 2008 Category: Biology Source Type: info

Assessing the Risk of Undesirable Immunogenicity/ Allergenicity of Plant-Derived Therapeutic ProteinsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Therapeutic proteins have an intrinsic potential to induce undesirable immune and allergic responses. The nature of the expression system and the control of the manufacturing process represent extrinsic factors that could modify this potential. Accordingly, regulatory agencies require sponsors to assess the risk of clinical immune and allergic responses that could be associated with the production of therapeutic proteins in transgenic plants. Since factors related to the clinical use of the product–including the immune status and genetic background of subjects–are also relevant, the risk assessment needs to bal...
Source: Springer protocols feed by Plant Sciences - June 1, 2008 Category: Biology Source Type: info

Cowpea Mosaic Virus-Based Systems for the Expression of Antigens and Antibodies in PlantsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
This chapter describes the use of Cowpea mosaic virus-based vectors for the production of foreign proteins such as antigens and antibodies in plants. The systems include vectors based on both full-length and deleted versions of RNA-2. In both cases, the modified RNA-2 is replicated by coinoculation with RNA-1. The constructs based on full-length RNA-2 retain the ability to spread systemically throughout an inoculated plant and the infection can be passaged. The vector based on a deleted version of RNA-2 can stably incorporate larger inserts but lacks the ability to move systemically. However, it has the added advantage of ...
Source: Springer protocols feed by Plant Sciences - June 1, 2008 Category: Biology Source Type: info

Biosafety, Risk Assessment and Regulation of Plant-Made PharmaceuticalsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The technology for plant-made pharmaceuticals (PMPs) has progressed significantly over the last few years, with the first commercial products for human use expected to reach the market by 2009 (see Note 1 ). As part of the ‘next generation’ of genetically modified (GM) crops, PMPs will be subject to additional biosafety considerations and are set to challenge the complex and overlapping regulations that currently govern GM plants, plant biologics (see Note 2 ) and ‘conventional’ pharmaceutical production. The areas of responsibility are being mapped out between the di...
Source: Springer protocols feed by Plant Sciences - June 1, 2008 Category: Biology Source Type: info

Transient Expression of Antibodies in Plants Using Syringe AgroinfiltrationEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The improvements in agroinfiltration methods for plant-based transient expression now allow the production of significant amounts of recombinant proteins in a matter of days. While vacuum-based agroinfiltration has been brought to large scale to meet the cost, speed and surge capacity requirements for vaccine and therapeutic production, the more accessible and affordable syringe agroinfiltration procedure still represents a fast and high-yielding approach to recombinant protein production at lab scale. The procedure exemplified here has proven its reproducibility and high-yield capacity for the production of proteins with ...
Source: Springer protocols feed by Plant Sciences - June 1, 2008 Category: Biology Source Type: info

Rapid System for Evaluating Bioproduction Capacity of Complex Pharmaceutical Proteins in PlantsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Transgene product yield remains a key limitation in commercializing plant-derived pharmaceutical proteins. Although significant progress has been made in understanding the roles of promoters, enhancers, integration sites, codon usage, cryptic RNA sites, silencing, and product compartmentalization on product yield and quality, researchers still cannot reliably predict which proteins will be produced at high levels or what manipulations will guarantee enhanced productivity. We have optimized a simple transient expression system in Nicotiana benthamiana enabling rapid assessment of transgene potential for plant-based bioprodu...
Source: Springer protocols feed by Plant Sciences - June 1, 2008 Category: Biology Source Type: info

Production and Localization of Recombinant Pharmaceuticals in Transgenic SeedsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Among the many plant-based production systems that have been developed for pharmaceutical proteins, seeds have the useful advantage of accumulating proteins in a relatively small volume, and recom-binant proteins are very stable in dry seeds allowing long-term storage and facilitating distribution before processing.
Source: Springer protocols feed by Plant Sciences - June 1, 2008 Category: Biology Source Type: info

Production of Antibody Fragments in Arabidopsis SeedsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Plants offer a number of attractive benefits over conventional mammalian or bacterial cell culture systems for the production of valuable pharmaceutical and industrial proteins. Currently, antibodies and their derived fragments represent the largest and most important group of biotechnological products in clinical trials. In particular, single-chain antibodies are an interesting class of biopharmaceuticals because they are able to overcome specific problems associated with full-length antibodies. Another valuable antibody format is the scFv-Fc: fusion of the Fc domain to a single-chain variable fragment restores antibody e...
Source: Springer protocols feed by Plant Sciences - June 1, 2008 Category: Biology Source Type: info

Production of Plantibodies in Nicotiana PlantsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Because of the wide use and high demand in medicine, monoclonal antibodies are among the main recombinant pharmaceuticals at present, although present limitations of the productive platforms for monoclonal antibodies are driving the improvement of the large-scale technologies and the development of alternative expression systems. This has drawn the attention on plants as expression system for monoclonal antibodies and related derivatives, owning the capacity of plants to properly express and process eukaryotic proteins with biological activity resembling that of the natural proteins. In this chapter, the procedures from th...
Source: Springer protocols feed by Plant Sciences - June 1, 2008 Category: Biology Source Type: info

Physcomitrella patens : A Non-Vascular Plant for Recombinant Protein ProductionEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The moss Physcomitrella patens is a long-standing model for studying plant development, growth and cell differentiation in particular. Interest in this non-vascular plant arose following the discovery that homologous recombination is an efficient process. P. patens is, therefore, a tool of choice not only to study gene function but also for recombinant protein production. This system has many attributes that are advantageous for molecular farming: protein production in cell suspension, the possibility of generating targeted knockout mutants for glycoengineering and quantitative optimization for protein production. In terms...
Source: Springer protocols feed by Plant Sciences - June 1, 2008 Category: Biology Source Type: info

Production of Recombinant Proteins in Suspension–Cultured Plant CellsEmail this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Plants have emerged in the past decade as a suitable alternative to the current production systems for recombinant pharmaceutical proteins and, today their potential for low-cost production of high quality, much safer and biologically active mammalian proteins is largely documented.
Source: Springer protocols feed by Plant Sciences - June 1, 2008 Category: Biology Source Type: info