Lentivirus Gene Engineering Protocols

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Lentivirus Gene Engineering Protocols

Maurizio Federico
Lentivirus Gene Engineering Protocols Book PDF

Author : Maurizio Federico
Publisher : Springer Science & Business Media
Page : 310 pages
File Size : 44,7 Mb
Release : 2008-02-03
Category : Science
ISBN : 9781592593934

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Lentivirus Gene Engineering Protocols by Maurizio Federico Pdf

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

Maurizio Federico
Lentiviral Vectors and Exosomes as Gene and Protein Delivery Tools Book PDF

Author : Maurizio Federico
Publisher : Humana
Page : 276 pages
File Size : 49,7 Mb
Release : 2018-05-31
Category : Medical
ISBN : 1493981293

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Lentiviral Vectors and Exosomes as Gene and Protein Delivery Tools by Maurizio Federico Pdf

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

Didier Trono
Lentiviral Vectors Book PDF

Author : Didier Trono
Publisher : Springer Science & Business Media
Page : 276 pages
File Size : 54,8 Mb
Release : 2001-12-14
Category : Medical
ISBN : 3540421904

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Lentiviral Vectors by Didier Trono Pdf

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

Sumitra Deb,Swati Palit Deb
p53 Protocols Book PDF

Author : Sumitra Deb,Swati Palit Deb
Publisher : Springer Science & Business Media
Page : 282 pages
File Size : 41,8 Mb
Release : 2008-02-02
Category : Science
ISBN : 9781592594085

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p53 Protocols by Sumitra Deb,Swati Palit Deb Pdf

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

Barry S. Selinsky
Membrane Protein Protocols Book PDF

Author : Barry S. Selinsky
Publisher : Springer Science & Business Media
Page : 330 pages
File Size : 45,9 Mb
Release : 2008-02-03
Category : Science
ISBN : 9781592594009

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Membrane Protein Protocols by Barry S. Selinsky Pdf

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

John M. S. Bartlett,David Stirling
PCR Protocols Book PDF

Author : John M. S. Bartlett,David Stirling
Publisher : Springer Science & Business Media
Page : 1083 pages
File Size : 47,9 Mb
Release : 2008-02-03
Category : Science
ISBN : 9781592593842

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PCR Protocols by John M. S. Bartlett,David Stirling Pdf

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.

Inflammation Protocols

Paul G. Winyard,Derek A. Willoughby
Inflammation Protocols Book PDF

Author : Paul G. Winyard,Derek A. Willoughby
Publisher : Springer Science & Business Media
Page : 373 pages
File Size : 52,5 Mb
Release : 2008-02-03
Category : Medical
ISBN : 9781592593743

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Inflammation Protocols by Paul G. Winyard,Derek A. Willoughby Pdf

Inflammation has been described as the basis of many pathologies of human disease. When one considers the updated signs of inflammation, they would be vasodilation, cell migration, and, in the case of chronic inflam- tion, cell proliferation, often with an underlying autoimmune basis. Gen- ally, inflammation may be divided into acute, chronic, and autoimmune, - though the editors believe that most, if not all, chronic states are often the result of an autoimmune response to an endogenous antigen. Thus, a proper understanding of the inflammatory basis may provide clues to new therap- tic targets not only in classical inflammatory diseases, but atherosclerosis, cancer, and ischemic heart disease as well. The lack of advances in classical inflammatory diseases, such as rh- matoid arthritis, may in part arise from a failure to classify the disease into different forms. That different forms exist is exemplified in patients with d- fering responses to existing antiinflammatory drugs, ranging from nonresponders to very positive responders for a particular nonsteroidal an- inflammatory drug (NSAID). Though researchers have progressively unr- eled the mechanisms, the story is far from complete. It should also be noted that the inflammatory response is part of the innate immune response, or to use John Hunter’s words in 1795, “inflammation is a salutary response.” That may be applied in particular to the defensive response to invading micro- ganisms.

Protein Kinase C Protocols

Alexandra C. Newton
Protein Kinase C Protocols Book PDF

Author : Alexandra C. Newton
Publisher : Springer Science & Business Media
Page : 565 pages
File Size : 43,8 Mb
Release : 2008-02-03
Category : Science
ISBN : 9781592593972

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Protein Kinase C Protocols by Alexandra C. Newton Pdf

Since the discovery that protein kinase C (PKC) transduces the ab- dance of signals that result in phospholipid hydrolysis, this enzyme has been at the forefront of research in signal transduction. Protein Kinase C Protocols covers fundamental methods for studying the structure, function, regulation, subcellular localization, and macromolecular interactions of PKC. Protein Kinase C Protocols is divided into 11 sections representing the major aspects of PKC regulation and function. Part I contains an introduction and a historical perspective on the discovery of PKC by Drs. Yasutomi Nishizuka and Ushio Kikkawa. Part II describes methods to purify PKC. Part III describes the standard methods for measuring PKC activity: its enzymatic activity and its stimulus-dependent translocation from the cytosol to the membrane. Part IV describes methods for measuring the membrane interaction of PKC in vivo and in vitro. Part V provides methodologies and techniques for measuring the ph- phorylation state of PKC, including a protocol for measuring the activity of PKC’s upstream kinase, PDK-1. Novel methods for identifying substrates are described in Part VI. Part VII presents protocols for expressing and analyzing the membrane targeting domains of PKC. Part VIII provides a comprehensive c- pilation of methods used to identify binding partners for PKC. Part IX describes pharmacological probes used to study PKC. The book ends with a presentation of genetic approaches to study PKC (Part X) and a discussion of approaches used to study PKC in disease (Part XI).

Protein Misfolding and Disease

Peter Bross,Niels Gregersen
Protein Misfolding and Disease Book PDF

Author : Peter Bross,Niels Gregersen
Publisher : Springer Science & Business Media
Page : 317 pages
File Size : 43,8 Mb
Release : 2008-02-02
Category : Science
ISBN : 9781592593941

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Protein Misfolding and Disease by Peter Bross,Niels Gregersen Pdf

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

Daniele D’Ambrosio,Francesco Sinigaglia
Cell Migration in Inflammation and Immunity Book PDF

Author : Daniele D’Ambrosio,Francesco Sinigaglia
Publisher : Springer Science & Business Media
Page : 283 pages
File Size : 42,8 Mb
Release : 2008-02-02
Category : Medical
ISBN : 9781592594351

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Cell Migration in Inflammation and Immunity by Daniele D’Ambrosio,Francesco Sinigaglia Pdf

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.

E. coli Plasmid Vectors

Nicola Casali,Andrew Preston
E coli Plasmid Vectors Book PDF

Author : Nicola Casali,Andrew Preston
Publisher : Springer Science & Business Media
Page : 311 pages
File Size : 47,6 Mb
Release : 2008-02-03
Category : Science
ISBN : 9781592594092

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E. coli Plasmid Vectors by Nicola Casali,Andrew Preston Pdf

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.

Plant Functional Genomics

Erich Grotewold
Plant Functional Genomics Book PDF

Author : Erich Grotewold
Publisher : Springer Science & Business Media
Page : 443 pages
File Size : 49,7 Mb
Release : 2008-02-03
Category : Science
ISBN : 9781592594139

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Plant Functional Genomics by Erich Grotewold Pdf

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.

Atomic Force Microscopy

Pier Carlo Braga,Davide Ricci
Atomic Force Microscopy Book PDF

Author : Pier Carlo Braga,Davide Ricci
Publisher : Springer Science & Business Media
Page : 388 pages
File Size : 43,7 Mb
Release : 2008-02-02
Category : Science
ISBN : 9781592596478

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Atomic Force Microscopy by Pier Carlo Braga,Davide Ricci Pdf

The natural, biological, medical, and related sciences would not be what they are today without the microscope. After the introduction of the optical microscope, a second breakthrough in morphostructural surface analysis occurred in the 1940s with the development of the scanning electron microscope (SEM), which, instead of light (i. e. , photons) and glass lenses, uses electrons and electromagnetic lenses (magnetic coils). Optical and scanning (or transmission) electron microscopes are called “far-field microscopes” because of the long distance between the sample and the point at which the image is obtained in comparison with the wavelengths of the photons or electrons involved. In this case, the image is a diffraction pattern and its resolution is wavelength limited. In 1986, a completely new type of microscopy was proposed, which, without the use of lenses, photons, or electrons, directly explores the sample surface by means of mechanical scanning, thus opening up unexpected possibilities for the morphostructural and mechanical analysis of biological specimens. These new scanning probe microscopes are based on the concept of near-field microscopy, which overcomes the problem of the limited diffraction-related resolution inherent in conventional microscopes. Located in the immediate vicinity of the sample itself (usually within a few nanometers), the probe records the intensity, rather than the interference signal, thus significantly improving resolution. Since the most we- known microscopes of this type operate using atomic forces, they are frequently referred to as atomic force microscopes (AFMs).

Directed Enzyme Evolution

Frances H. Arnold,George Georgiou
Directed Enzyme Evolution Book PDF

Author : Frances H. Arnold,George Georgiou
Publisher : Springer Science & Business Media
Page : 381 pages
File Size : 41,6 Mb
Release : 2008-02-02
Category : Science
ISBN : 9781592593965

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Directed Enzyme Evolution by Frances H. Arnold,George Georgiou Pdf

Directed evolution comprises two distinct steps that are typically applied in an iterative fashion: (1) generating molecular diversity and (2) finding among the ensemble of mutant sequences those proteins that perform the desired fu- tion according to the specified criteria. In many ways, the second step is the most challenging. No matter how cleverly designed or diverse the starting library, without an effective screening strategy the ability to isolate useful clones is severely diminished. The best screens are (1) high throughput, to increase the likelihood that useful clones will be found; (2) sufficiently sen- tive (i. e. , good signal to noise) to allow the isolation of lower activity clones early in evolution; (3) sufficiently reproducible to allow one to find small improvements; (4) robust, which means that the signal afforded by active clones is not dependent on difficult-to-control environmental variables; and, most importantly, (5) sensitive to the desired function. Regarding this last point, almost anyone who has attempted a directed evolution experiment has learned firsthand the truth of the dictum “you get what you screen for. ” The protocols in Directed Enzyme Evolution describe a series of detailed p- cedures of proven utility for directed evolution purposes. The volume begins with several selection strategies for enzyme evolution and continues with assay methods that can be used to screen enzyme libraries. Genetic selections offer the advantage that functional proteins can be isolated from very large libraries s- ply by growing a population of cells under selective conditions.

G Protein Signaling

Alan V. Smrcka
G Protein Signaling Book PDF

Author : Alan V. Smrcka
Publisher : Springer Science & Business Media
Page : 246 pages
File Size : 55,8 Mb
Release : 2008-02-02
Category : Science
ISBN : 9781592594306

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G Protein Signaling by Alan V. Smrcka Pdf

Alan V. Smrcka presents a collection of cutting-edge methods for investigating G protein signaling from a variety of perspectives ranging from in vitro biochemistry to whole animal studies. Among the readily reproducible techniques presented are those for the purification of G proteins and effectors enzymes, assays of these purified G proteins and effector enzymes, and for the study of G proteins interactions with effectors in intact cells. Additional methods are provided for assaying G protein coupled receptor structure, function, and localization, and for studying the physiological roles for endogenous G proteins.