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Lentivirus Gene Engineering Protocols
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 inv...
Membrane Transporters
Studies of membrane transporters have had great impact on our und- standing human diseases and the design of effective drugs. About 30% of current clinically marketed drugs are targeting membrane transporters or channels. Membrane Transporters: Methods and Protocols provides various practical methodologies for the ongoing research on membrane transporters. To provide readers the most up-to-date information, several emerging fields and methodologies are embraced in this book, including pharmacogenomics, bioin-formatics, and microarray technology. Pharmacogenomics studies of membrane transporters are useful in drug discovery and in predicting drug responses in the clinic. In this volume, the c...
Capillary Electrophoresis of Carbohydrates
A collection of cutting-edge techniques for using capillary electrophoresis (CE) to analyze complex carbohydrates. These readily reproducible protocols provide methods for sample preparation, analysis of mono- and oligosaccharides, glycoproteins, and glycoconjugates. A useful appendix describes the structures of the most commonly encountered carbohydrate residues and olgosaccharides from mammalian and bacterial origins. Each protocol contains detailed information on reagents, apparatus, notes, comments, and tips on procedures.
Directed Enzyme Evolution
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 c...
Cell Migration in Inflammation and Immunity
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, angio...
PCR Protocols
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.
Tumor Suppressor Genes
It has become clear that tumors result from excessive cell proliferation and a corresponding reduction in cell death caused by the successive accumulation of mutations in key regulatory target genes over time. During the 1980s, a number of oncogenes were characterized, whereas from the 1990s to the present, the emp- sis has shifted to tumor suppressor genes (TSGs). It has become clear that oncogenes and TSGs function in the same pathways, providing positive and negative growth regulatory activities. The signaling pathways controlled by these genes involve virtually every process in cell biology, including nuclear events, cell cycle, cell death, cytoskeletal, cell membrane, angiogenesis, and ...
Inflammation Protocols
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 disea...
Plant Functional Genomics
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...
Functional Genomics
This collection of robust, readily reproducible methods for microarray-based studies includes expert guidance in the optimal data analysis and informatics. On the methods side are proven techniques for monitoring subcellular RNA localization en masse, for mapping chromosomes at the resolution of a single gene, and for surveying the steady-state genome-wide distribution of DNA binding proteins in vivo. For those workers dealing with massive data sets, the book discusses the methodological aspects of data analysis and informatics in the design of microarray experiments, the choice of test statistic, and the assessment of observational significance, data reduction, and clustering.
