Lentivirus Gene Engineering Protocols


Book Description

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.




Gastrointestinal Physiology and Diseases


Book Description

This volume provides a comprehensive collection of classical and cutting edge protocols and techniques to examine the normal development and physiological functions of the gastrointestinal system and to model the most common digestive diseases. The chapters focus on diverse research topics including ex vivo systems to study gastrointestinal development and functions, in vivo imaging of the gastrointestinal tract, isolation and characterization of intestinal immune cells, and animal models of gastrointestinal inflammation and cancer. The Gastrointestinal Physiology and Diseases: Methods and Protocols book targets wide audience of physiologists, cell and developmental biologists, immunologists, and physician-scientists working in the field of gastroenterology and beyond. 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. Highly practical and clearly written, Gastrointestinal Physiology and Diseases: Methods and Protocols will serve both seasoned researchers as well as newcomers to the field and will provide a unique resource and expert guidance to modern laboratory techniques developed for examining normal functions and diseases of the gastrointestinal tract.




Lentiviral Vectors


Book Description

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.




PCR Protocols


Book Description

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.




p53 Protocols


Book Description

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.




Human Embryonic Stem Cells


Book Description

Since the first successful isolation and cultivation of human embryonic stem cells at the University of Wisconsin, Madison in 1998, there has been high levels of both interest and controversy in this area of research. This book provides a concise overview of an exciting field, covering the characteristics of both human embryonic stem cells and pluripotent stem cells from other human cell lineages. The following chapters describe state-of-the-art differentiation and characterization of specific ectoderm, mesoderm and endoderm-derived lineages from human embryonic stem cells, emphasizing how these can be used to study human developmental mechanisms. A further chapter discusses genetic manipulation of human ES cells. The concluding section covers therapeutic applications of human ES cells, as well as addressing the ethical and legal issues that this research have raised.




Membrane Protein Protocols


Book Description

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.




Cytokine Protocols


Book Description

A collection of biochemical, cellular, and molecular techniques for unraveling and quantifying the events occurring between the initial contact of a cytokine at the membrane receptor and the eventual activation of gene transcription. The techniques used include the generation of transfectants, the immunohistochemical detection of cytokines in tissue sections, and optimized staining for cytoplasmic detection. Highlights include RT-PCR of small amounts of mRNA, in situ hybridization, biosensor analysis, measurement of biological activities and standardization, immunohistochemical and single-cell detection, and receptor isolation, characterization, and crystallization. Enjoy a quick and smooth introduction to the key methods used in cytokine research Use readily reproducible techniques that ensure successful experimental results Employ antisense-RNA, RT-PCR of small amounts of mRNA, and in situ hybridization.




Inflammation Protocols


Book Description

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


Book Description

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).