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Lentivirus Gene Engineering Protocols

Lentivirus Gene Engineering Protocols
Author: Maurizio Federico
Publisher: Springer Science & Business Media
Total Pages: 310
Release: 2008-02-03
Genre: Science
ISBN: 1592593933

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Cell gene engineering is emerging as a field with outstanding impact, not only in medicine/biology, but also, and perhaps most importantly, in agriculture and in all those food sciences involved in the fight against world hunger. Lentivirus vector-based technologies represent the last frontier in the development of powerful and reliable methods for both in vitro and in vivo gene transfer in eukaryotic animal cells. Although the design of lentivirus vectors is closely reminiscent of those already successfully applied to the construction of oncoretroviral vectors, some unique features, e.g., the ef- ciency in transducing both postmitotic and stem cells, render the use of lentivirus vectors invaluable. It has been a great pleasure to edit Lentivirus Gene Engineering Pro- cols, owing in part to the high level of enthusiasm that the authors dem- strated in contributing to this book. The fact that so many outstanding scientists engaged in lentivirus vector research have provided articles renders it so- thing more than a technical handbook. In addition to detailed descriptions of the most innovative methodologies, the reader may find very informative ov- views concerning both theoretical and practical aspects of the origin and the development of diverse lentivirus vector types. This, in my opinion, rep- sents a unique added value of this volume, which should help our work resist the passage of time, to which books such as this are particularly sensitive.


Lentiviral Vectors and Exosomes as Gene and Protein Delivery Tools

Lentiviral Vectors and Exosomes as Gene and Protein Delivery Tools
Author: Maurizio Federico
Publisher: Humana
Total Pages: 276
Release: 2018-05-31
Genre: Medical
ISBN: 9781493981298

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This volume provides current methods and protocols for gene and protein delivery based on both lentivirus-generated and spontaneously released nanovesicles. Lentiviral Vectors and Exosomes as Gene and Protein Delivery Tools guides readers through methods on macromolecule delivery and chapters describe the LV-based protocols of gene engineering. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Lentiviral Vectors and Exosomes as Gene and Protein Delivery Tools aims to ensure successful results in the further study of this vital field.


Lentiviral Vectors

Lentiviral Vectors
Author: Didier Trono
Publisher: Springer Science & Business Media
Total Pages: 276
Release: 2001-12-14
Genre: Medical
ISBN: 9783540421900

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For the first time a compilation of chapters that depict the biological bases underlying the development of lentiviral vectors, the techniques involved in the manufacture of this new gene delivery tool, and its most promising applications.


p53 Protocols

p53 Protocols
Author: Sumitra Deb
Publisher: Springer Science & Business Media
Total Pages: 282
Release: 2008-02-02
Genre: Science
ISBN: 1592594085

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Since the discovery of p53 as a tumor suppressor, numerous methods have evolved to reveal the unique structural features and biochemical functions of this protein. Several unique properties of p53 posed a challenge to understa- ing its normal function in the initial phase of its research. The low levels of p53 in normal cells, its stabilization under situations of genotoxic stress, induction of growth arrest, and apoptosis with stabilization of the protein, obstructed the visibility of its normal, unmutated function. The property of p53 that can sense a promoter and transactivate or inhibit is still not well understood. It is still not known whether it is the absence of the protein that causes tumorigenesis, or if its mutants have a dominant role in inducing cancer. p53 Protocols comprises eighteen chapters for the study of the diverse properties of p53 and related proteins. The methods included are invaluable for delineating the function of other proteins that may function as tumor suppr- sors or growth suppressors. The chapters are not presented in any schematic order, for the importance and diversity of the functions of p53 make it imp- sible to organize them suitably. We have made a sincere effort to collect the methods most useful to those investigators working on tumor suppressors or growth suppressors. The purpose of p53 Protocols is not only to provide investigators with methods to analyze similar biochemical functions, but also to familiarize them with the associated problems that arose during the course of investigations.


Membrane Protein Protocols

Membrane Protein Protocols
Author: Barry S. Selinsky
Publisher: Springer Science & Business Media
Total Pages: 330
Release: 2008-02-03
Genre: Science
ISBN: 159259400X

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Knowledge of the three-dimensional structure of a protein is absolutely required for the complete understanding of its function. The spatial orientation of amino acids in the active site of an enzyme demonstrates how substrate specificity is defined, and assists the medicinal chemist in the design of s- cific, tight-binding inhibitors. The shape and contour of a protein surface hints at its interaction with other proteins and with its environment. Structural ana- sis of multiprotein complexes helps to define the role and interaction of each individual component, and can predict the consequences of protein mutation or conditions that promote dissociation and rearrangement of the complex. Determining the three-dimensional structure of a protein requires milligram quantities of pure material. Such quantities are required to refine crystallization conditions for X-ray analysis, or to overcome the sensitivity limitations of NMR spectroscopy. Historically, structural determination of proteins was limited to those expressed naturally in large amounts, or derived from a tissue or cell source inexpensive enough to warrant the use of large quantities of cells. H- ever, with the advent of the techniques of modern gene expression, many p- teins that are constitutively expressed in minute amounts can become accessible to large-scale purification and structural analysis.


PCR Protocols

PCR Protocols
Author: John M. S. Bartlett
Publisher: Springer Science & Business Media
Total Pages: 1083
Release: 2008-02-03
Genre: Science
ISBN: 1592593844

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In this new edition, the editors have thoroughly updated and dramatically expanded the number of protocols to take advantage of the newest technologies used in all branches of research and clinical medicine today. These proven methods include real time PCR, SNP analysis, nested PCR, direct PCR, and long range PCR. Among the highlights are chapters on genome profiling by SAGE, differential display and chip technologies, the amplification of whole genome DNA by random degenerate oligonucleotide PCR, and the refinement of PCR methods for the analysis of fragmented DNA from fixed tissues. Each fully tested protocol is described in step-by-step detail by an established expert in the field and includes a background introduction outlining the principle behind the technique, equipment and reagent lists, tips on trouble shooting and avoiding known pitfalls, and, where needed, a discussion of the interpretation and use of results.


Protein Misfolding and Disease

Protein Misfolding and Disease
Author: Peter Bross
Publisher: Springer Science & Business Media
Total Pages: 317
Release: 2008-02-02
Genre: Science
ISBN: 1592593941

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For decades it has been known that structured conformations are important for the proper functioning of most cellular proteins. However, appreciation that protein folding to the functional conformations as well as the structural maintenance of protein molecules are very complex processes has only emerged during the last ten years. The intimate interplay uncovered by this scientific development led us to realize that perturbations of the protein folding process and disturbances of conformational maintenance are major disease mechanisms. This development has given rise to the concept of conformational diseases and the broader signature of protein folding diseases, comprising diseases in which mutations or environmental stresses may result in a partial misfolding that leads then to alternative conformations capable of disturbing cellular processes. This may happen by self-association (aggregation), as in prion and Alzheimer’s diseases, or by incorporation of alternatively folded subunits into structural entities, as in collagen diseases. Another possibility is that folding to the native structure is impaired or abolished, resulting in decreased stea- state levels of the correctly folded protein, as is observed in cystic fibrosis and 1-antitrypsin deficiency, as well as in many enzyme deficiencies. In addition, deficiencies of proteins that are engaged in assisting and supervising protein folding (protein quality control) may impair the folding of many other proteins, resulting in pathological phenotypes. Examples of this are the spastic paraplegia attributable to mutations in mitochondrial protease/chaperone complexes.


Cell Migration in Inflammation and Immunity

Cell Migration in Inflammation and Immunity
Author: Daniele D’Ambrosio
Publisher: Springer Science & Business Media
Total Pages: 283
Release: 2008-02-02
Genre: Medical
ISBN: 1592594352

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Chemokines and their receptors play a central role in the pathogenesis of numerous, perhaps all, acute and chronic inflammatory diseases. About 50 distinct chemokines produced by a variety cell types and tissues either c- stitutively or in response to inflammatory stimuli are involved in a plethora of biological processes. These small secreted proteins exert their exquisitely variegated functions upon binding to a family of seven-transmembrane spanning G-protein coupled receptors (GPCRs) composed of almost 20 distinct entities. The biological activities of chemokines range from the control of leukocyte trafficking in basal and inflammatory conditions to the regulation of hema- poiesis, angiogenesis, tissue architecture, and organogenesis. The basis for such diversified activities rests, on one hand, upon the ubiquitous nature of chemokine production and chemokine receptor expression. Virtually every cell type can produce chemokines and expresses a unique combination of chemokine receptors. On the other hand, chemokine receptors make use of a flexible and complex network of intracellular signaling machineries that can regulate a variety of cellular functions ranging from cell migration, growth, and differentiation to death. As knowledge of the size of chemokine and chemokine receptor families rapidly reaches completeness, much is still to be uncovered in terms of fu- tional architecture of the chemokine system. The disparity between the large number of chemokines and that smaller number of receptors is balanced by the promiscuity in ligand–receptor interactions, with multiple chemokines binding to the same receptor and several chemokines binding to more than one receptor.


Plant Functional Genomics

Plant Functional Genomics
Author: Erich Grotewold
Publisher: Springer Science & Business Media
Total Pages: 443
Release: 2008-02-03
Genre: Science
ISBN: 1592594131

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Functional genomics is a young discipline whose origin can be traced back to the late 1980s and early 1990s, when molecular tools became available to determine the cellular functions of genes. Today, functional genomics is p- ceived as the analysis, often large-scale, that bridges the structure and organi- tion of genomes and the assessment of gene function. The completion in 2000 of the genome sequence of Arabidopsis thaliana has created a number of new and exciting challenges in plant functional genomics. The immediate task for the plant biology community is to establish the functions of the approximately 25,000 genes present in this model plant. One major issue that will remain even after this formidable task is c- pleted is establishing to what degree our understanding of the genome of one model organism, such as the dicot Arabidopsis, provides insight into the or- nization and function of genes in other plants. The genome sequence of rice, completed in 2002 as a result of the synergistic interaction of the private and public sectors, promises to significantly enrich our knowledge of the general organization of plant genomes. However, the tools available to investigate gene function in rice are lagging behind those offered by other model plant systems. Approaches available to investigate gene function become even more limited for plants other than the model systems of Arabidopsis, rice, and maize.


E. coli Plasmid Vectors

E. coli Plasmid Vectors
Author: Nicola Casali
Publisher: Springer Science & Business Media
Total Pages: 311
Release: 2008-02-03
Genre: Science
ISBN: 1592594093

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A comprehensive collection of readily reproducible techniques for the manipulation of recombinant plasmids using the bacterial host E. coli. The authors describe proven methods for cloning DNA into plasmid vectors, transforming plasmids into E. coli, and analyzing recombinant clones. They also include protocols for the construction and screening of libraries, as well as specific techniques for specialized cloning vehicles, such as cosmids, bacterial artificial chromosomes, l vectors, and phagemids. Common downstream applications such as mutagenesis of plasmids, recombinant protein expression, and the use of reporter genes, are also described.