This volume presents state-of-the art methods for the synthesis, design, assembly, post synthesis processing, and application of synthetic DNA to modern biotechnology. Chapters are divided into three general sections focusing on protocols for the computational design of synthetic DNA sequences, the synthesis, assembly and cloning of synthetic DNA, and post-synthesis error reduction strategies. 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, Synthetic DNA: Methods and Protocols aims to help researchers further their research on manipulate DNA sequences.

This book explores the major cytokines, such as IL-1 and IFN- , with respect to the regulation of their gene expression and protein production in specific immune cell types. It discusses both healthy physiological settings and in pathological situations in which the expression of some cytokines could be dysregulated, resulting in either immunodeficiency or exacerbated inflammatory sequelae in animal models as well as in human patients. Cytokines are important regulators of immune responses that require the highly coordinated participation and communication of multiple cell types. The expression of cytokines by various producer cell types is therefore carefully regulated in response to environmental cues at multiple levels: transcription, translation and posttranslational modification. Presenting cutting-edge advances in our understanding of the regulation of cytokine expression, this book is a valuable resource for anyone involved or interested in immune regulation.

In this book, leading figures in the field of Developmental Origins of Health and Disease provide up-to-date information from human clinical trials, cohorts, and animal physiology experiments to reveal the interdependence between parental obesity and health of the offspring. Obesity of the mother and father produces obesity in their offspring, so we are caught up in an intergenerational cycle, which means that even our children's future health is in peril. This book gives a timely and much-needed synthesis of the mechanisms, potential targets of future interventions, and the challenges that need to be overcome in order to break the intergenerational cycle of obesity. This has profound implications for the way in which scientific, clinical and health policy activities are to be directed in order to combat the so-called epidemic of obesity, as well as diabetes, cancer and cardiovascular disease. The book will be of interest to students, clinicians, researchers and health policy makers who are either seeking an introduction to the area of Developmental Origins of Health and Disease or have a specific interest in the pathogenesis of obesity.

This updated book includes meiosis methods ranging from classical genetic approaches with budding yeast to high resolution microscopy and computational methods for the analysis of recombination and modeling gene expression networks. Cutting-edge procedures for the analysis of double strand breaks at single nucleotide resolution, analysis of translation by ribosome profiling, the use of fluorescent markers to analyze recombination, and strategies for the use of conditional expression to study chromatin protein dynamics are detailed. Advanced cytology methods for live and fixed cell microscopy and image analysis for yeast, drosophila, and mouse are also included. Written for the highly successful Methods in Molecular Biology series, 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. Detailed and practical, Meiosis, Second Edition will prove to be invaluable to biologists, geneticists, biochemists, and anyone investigating meiosis, recombination, and cellular differentiation.

This volume is divided in four sections; covering genome wide approaches, techniques for characterize of paRNA structural features are described, selecting pa-RNA, and paRNA therapeutic potential. Chapters describe how siRNAsdirected against paRNAs can be applied in vivo to modulate transcription of important genes controlled by paRNAs. 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, Promoter Associated RNA: Methods and Protocols aims to demonstrate paRNAs as new class of regulatory molecules, to further investigate and value as tools for fine transcriptional tuning.

This volume describes a variety of protocols that will allow the readers to study different aspects of transcriptional and posttranscriptional gene expression regulation in eukaryotic cells. Chapters focus on the latest use of CRISPRi and RNAi technologies for studying various aspects of transcriptional and posttranscriptional regulation and tools to navigate protocols on key bioinformatics. 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, Eukaryotic Transcription and Post-Transcription Gene Expression Regulation aims to ensure successful results in the further study of this vital field.

This manual offers detailed protocols for fluorescence in situ hybridization (FISH) and comparative genomic hybridization approaches, which have been successfully used to study various aspects of genomic behavior and alterations. Methods using different probe and cell types, tissues and organisms, such as mammalians, fish, amphibians (including lampbrush-chromosomes), insects, plants and microorganisms are described in 57 chapters. In addition to multicolor FISH procedures and special applications such as the characterization of marker chromosomes, breakpoints, cryptic aberrations, nuclear architectures and epigenetic changes, as well as comparative genomic hybridization studies, this 2nd edition describes how FISH can be combined with other techniques. The latter include immunostaining, electron microscopy, single cell electrophoresis and microdissection. This well-received application guide provides essential protocols for beginning FISHers and FISH experts alike working in the fields of human genetics, microbiology, animal and plant sciences.

This book is about "Angiogenesis". A process in which new vasculature is formed from pre-existing capillaries. Angiogenesis process is associated with the proliferation and growth of both physiologically normal and neoplastic tissues, through the formation of vascular supply, essential for delivering growth requirements such as oxygen and nutrients. The book describes more than 100 genes and their key regulatory functions in the context of normal healthy condition, disease and malignancy, cancer proliferation and progression. New insights into the role of angiogenesis and the therapeutic inhibition of its regulators are investigated, due to the great potential for exploitation in the development of a novel treatment for cancer. New scientists, junior researchers and biomedical science students will find this book an invaluable introductory reference to their insight about angiogenesis and angiogenic role of more than 100 angiogenes and their role in healthy, disease and malignant conditions.

Ribonucleic acid (RNA) binding proteins currently number in the thousands and defects in their function are at the heart of diseases such as cancer and neurodegeneration. RNA binding proteins have become implicated in the intricate control of surprisingly diverse biological settings, such as circadian rhythm, stem cell self-renewal, oncogenesis and germ cell development. This book surveys a range of genome-wide and systems approaches to studying RNA binding proteins, the importance of RNA binding proteins in development, cancer and circadian rhythm.

This volume explores a broad range of different genotyping techniques. Genotyping: Methods and Protocols consists of chapters that cover numerous topics such as: an overview of multiplexed microsatellite analysis; High Resolution Melt analysis and TaqMan-based assays; in situ analysis of variants in single RNA molecules; the MassARRAY system and Molecular Inversion Probes; Pulsed Field Gel Electrophoresis, Paralogue Ratio Test, and Multiplex Ligation-Dependent Probe Amplification; long-range PCR combined with PacBio sequencing; Targeted Locus Amplification; Multilocus Sequence Typing and rapid SNP detection with pyrosequencing; and genotyping-by-sequencing for plant analysis. Finally, the volume concludes with a summary of pertinent points to describe genetic variation. 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. Thorough and practical, Genotyping: Methods and Protocols is a valuable resource for anyone interested in learning more about the diverse field of genotyping.

The concept of immunotherapy was in infancy when the first edition was written; since then, major advances have been made, not only with several prominent clinical trials, but also with the approval of cell-based therapy by the FDA for the treatment of cancer in 2010. These events resulted in a gradually narrowing gap between early scientific knowledge and the late development of immune-based therapies. Consequently, the significance and magnitude of these advances warranted a revision of this contribution; this revised edition will provide a deeper understanding of the recent advances and discoveries related to the function of the immune response and their applications in the development of novel therapies to treat human diseases. Some of the key discoveries during the past five years include: the identification of the new subsets of helper T cells; new cytokines and their networks; and novel signal transduction mechanisms. For example, the identification of TH17 subset of helper T cells, in addition to TH1 and TH2 cells, not only advanced our understanding of the function of the basic immune response, but also raised our awareness of the possible etiology and pathogenesis of diseases such as allergy, asthma, rheumatoid arthritis, and other auto-immune/immune system based diseases. The newly identified powerful cytokine networks, that regulate both innate and acquired immune responses, emerged as a result of the finding of new cell types such as innate lymphoid cells and iNKT. Identification of the novel cytokines and their networks has advanced our knowledge of the mechanisms involved in the maintenance of tissue homeostasis, including inflammation and tissue repair during stress and injury. The development of HIV vaccines has also seen dramatic changes over the last few years. There has been a shift from a sole focus on T cell vaccines to a holistic approach that pertains to the induction of both humoral and cellular elements. This entails the induction of antibodies - both binding and neutralizing - to prevent infection. The cellular vaccination produces a safety net of CD8+ T-cell responses to suppress the replication of the virus in the infected patients, and both of the effector arms are aided by helper T cells. From the perspective of clinical applications, significant advances have also been made in: oral immunotherapy for allergic disease, the possible treatment of HIV infection, the development of new monoclonal antibodies and their fragments to treat human diseases, and immune cell based therapies for cancer.

This volume brings together recent developments in quasispecies theory extended to variable environments and practical applications in elucidating viral dynamics and treatment designs. In particular, the existence of an error threshold in rugged fitness landscapes has opened the way to a new antiviral strategy termed lethal mutagenesis, which is now under intensive theoretical, experimental and clinical investigation. As such the book explains how an understanding of quasispecies dynamics within infected organisms has increased our knowledge of viral disease events. From a clinical perspective, population dynamics highlights important problems for viral disease control, such as the selection of drug-resistant mutants that often accompanies treatment failures, and suggests means of increasing the effectiveness of antiviral treatments. The book is intended for students and scientists interested in basic and applied aspects of biophysics, chemistry, biology, evolution and medical virology.

This book discusses the molecular, biological, pathological, and clinical aspects of melanoma, with special emphasis in the new concepts of melanoma genetics. A multidisciplinary group of experts in Genetics, Dermatology, Pathology, and Melanoma Medical Oncology contribute state-of-the-art knowledge in melanoma research and clinical management, not only exposing the current status of knowledge of the topics but also providing their personal experiences and ideas about the future and potential practical application of the genetic aspects of melanoma. During the last few years we have witnessed an impressive amount of discoveries in the field of melanoma genetics which have changed our approach in understanding the pathogenesis and treatment of this lethal disease. Genetics of Melanoma is a practical approach to melanoma genetic mechanisms and their application in the diagnosis and treatment of this malignancy. It is an essential source of updated information and a powerful tool for clinicians, pathologists, and basic scientists who wish to understand, apply, and investigate the multiple new aspects of melanoma genetics.

This volume provides a thorough overview of the Wilms' Tumour Gene (WT1). The book begins with three review chapters that cover the involvement of WT1 in pediatric cancer, kidney disease, and tissue development and homeostasis. The next few chapters discuss cell marking and lineage tracing, epicardial cell methodology, colony forming assays for bone marrow stem cells, angiogenesis assays and zebrafish tools. The next group of chapters explores the latest tools in genomics, molecular biology, and biochemistry. They discuss dissecting transcription factor function in cell free systems, ChiP seq, proteomics, RNA interactome, and multiphoton imaging of lipids, measuring the binding constants of protein-nucleic acid interactions, and bioinformatics approaches for analyzing Next Generation Sequence data. The final chapter discusses protocols for clinical trials for immune therapy using anti-WT1 peptides. 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. Practical and thorough, The Wilms' Tumour (WT1) Gene: Methods and Protocols is a valuable resource for anyone who is interested in the diverse methodologies used in WT1 research.

High throughput sequencing (HTS) technologies have conquered the genomics and epigenomics worlds. The applications of HTS methods are wide, and can be used to sequence everything from whole or partial genomes, transcriptomes, non-coding RNAs, ribosome profiling, to single-cell sequencing. Having such diversity of alternatives, there is a demand for information by research scientists without experience in HTS that need to choose the most suitable methodology or combination of platforms and to define their experimental designs to achieve their specific objectives. Field Guidelines for Genetic Experimental Designs in High-Throughput Sequencing aims to collect in a single volume all aspects that should be taken into account when HTS technologies are being incorporated into a research project and the reasons behind them. Moreover, examples of several successful strategies will be analyzed to make the point of the crucial features. This book will be of use to all scientist that are unfamiliar with HTS and want to incorporate such technologies to their research.

This volume presents detailed laboratory protocols for in vitro synthesis of mRNA with favorable properties, its introduction into cells by a variety of techniques, and the measurement of physiological and clinical consequences such as protein replacement and cancer immunotherapy. Synthetic techniques are described for structural features in mRNA that provide investigational tools such as fluorescence emission, click chemistry, photo-chemical crosslinking, and that produce mRNA with increased stability in the cell, increased translational efficiency, and reduced activation of the innate immune response. Protocols are described for clinical applications such as large-scale transfection of dendritic cells, production of GMP-grade mRNA, redirecting T cell specificity, and use of molecular adjuvants for RNA vaccines. 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 laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Synthetic mRNA: Production, Introduction into Cells, and Physiological Consequences is a valuable and cutting-edge resource for both laboratory investigators and clinicians interested in this powerful and rapidly evolving technology.

This volume details a valuable collection of protocols and reviews, such as emerging experimental and theoretical approaches. These approaches have resulted in a substantial improvement in the understanding of chromosome architecture. Chromosome Architecture: Methods and Protocols guides readers through cutting-edge interdisciplinary methods which allow for an understanding of architecture of chromosomes with exceptionally enhanced resolution, both in terms of space and time. 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, Chromosome Architecture: Methods and Protocols aims to ensure successful results in the further study of this vital field.

This comprehensive volume explores human genetic engineering its pre-clinical and clinical applications, current developments, and as treatment for hereditary diseases. It presents and evaluates the most recent advances in the understanding of mammalian host DNA repair mechanisms, such as double-strand break induced gene targeting and mutagenesis, the development of zinc-finger nucleases, genome editing for neuromuscular diseases, phase integrases, triplex forming oligonucleotides and peptide nucleic acids, aptamer-guided gene targeting, AAV gene editing via DSB repair, engineered nucleases and trinucleotide repeat diseases, and creation of HIV-resistant cells. The expertly authored chapters contextualize current developments within the history of genome editing while also discussing the current and potential safety concerns of this rapidly growing field. Genome Editing: The Next Step in Gene Therapy, the latest volume in the American Society of Gene and Cell Therapy series, deftly illuminates the potential of genetic engineering technology to eradicate today's deadliest and most prolific diseases. It is ideal reading for clinicians and researchers in genetics and immunology.

This volume explores the epigenetic alterations and their association with various human cancers. Considering one of human cancer as an example, individual chapters are focused on defining the role of epigenetic regulators and underlying mechanisms in cancer growth and progression. Epigenetic alteration including DNA methylation, histone modification, nucleosome positioning and non-coding RNAs expression are involved in a complex network of regulating expression of oncogenes and tumor suppressor genes and constitute an important event of the multistep process of carcinogenesis. Recent advances in the understanding of the epigenetic regulation and detailed information of these epigenetic changes in various cancers provide new avenues of advancements in diagnostics, prognostics, and therapies of this highly fatal disease.

Computational Epigenetics and Diseases, written by leading scientists in this evolving field, provides a comprehensive and cutting-edge knowledge of computational epigenetics in human diseases. In particular, the major computational tools, databases, and strategies for computational epigenetics analysis, for example, DNA methylation, histone modifications, microRNA, noncoding RNA, and ceRNA, are summarized, in the context of human diseases. This book discusses bioinformatics methods for epigenetic analysis specifically applied to human conditions such as aging, atherosclerosis, diabetes mellitus, schizophrenia, bipolar disorder, Alzheimer disease, Parkinson disease, liver and autoimmune disorders, and reproductive and respiratory diseases. Additionally, different organ cancers, such as breast, lung, and colon, are discussed. This book is a valuable source for graduate students and researchers in genetics and bioinformatics, and several biomedical field members interested in applying computational epigenetics in their research.

This volume assembles a broad spectrum of methods used in long non-coding RNAs (lncRNA) research, ranging from computational annotation of lncRNA genes to molecular and cellular analyses of the function of individual lncRNA. Long Non-Coding RNAs: Methods and Protocols also discusses methods used to study circular RNAs and RNA splicing, as well as influential findings on lncRNA in human diseases. 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. Thorough and cutting-edge, Long Non-Coding RNAs: Methods and Protocols is a must-have for molecular biologists, cell and developmental biologists, specialists who conduct disease-oriented research, and bioinformatics experts who seek a better understanding on lncRNA expression and function by computational analysis of the massive sequencing data that are rapidly accumulating in recent years.

The aim of this book is to improve pyrosequencing protocols as well as instrumentation for better clinical use by describing improvements and novel applications of pyrosequencing technology. Divided into five parts, the book's thirty chapters explore advances in pyrosequencing template preparation, pyrosequencing technology innovations, multiplex pyrosequencing based on barcodes, the miniaturization of pyrosequencing equipment, as well as various applications. As part of the Springer Protocols program, chapters contain the kind of detail and practical implementation advice to guarantee successful results in the lab. Comprehensive and thorough, Advances and Clinical Practice in Pyrosequencing serves as a valuable reference for researchers who are engaged in personalized medicine, disease control, and DNA diagnosis in numerous other fields.

Every time a cell divides, a copy of its genomic DNA has to be faithfully copied to generate new genomic DNA for the daughter cells. The process of DNA replication needs to be precisely regulated to ensure that replication of the genome is complete and accurate, but that re-replication does not occur. Errors in DNA replication can lead to genome instability and cancer. The process of replication initiation is of paramount importance, because once the cell is committed to replicate DNA, it must finish this process. A great deal of progress has been made in understanding how DNA replication is initiated in eukaryotic cells in the past ten years, but this is the first one-source book on these findings. The Initiation of DNA Replication in Eukaryotes will focus on how DNA replication is initiated in eukaryotic cells. While the concept of replication initiation is simple, its elaborate regulation and integration with other cell processes results in a high level of complexity. This book will cover how the position of replication initiation is chosen, how replication initiation is integrated with the phases of the cell cycle, and how it is regulated in the case of damage to DNA. It is the cellular protein machinery that enables replication initiation to be activated and regulated. We now have an in-depth understanding of how cellular proteins work together to start DNA replication, and this new resource will reveal a mechanistic description of DNA replication initiation as well.

This book examines how post-transcriptional mechanisms control endocrine function. This includes newly identified regulatory mechanisms involved in hormone biosynthesis, control of hormone receptors and the outputs of hormone mediated signal transduction. Chapters address endocrine hormones including protein peptide/peptide, steroid, and non-steroidal hormones. The impacts of these mechanisms on disease and health are covered, providing a novel update to the scientific literature. Post-transcriptional regulatory mechanisms play an essential role in controlling dynamic gene expression. The outcome of this regulation includes control of the amount, timing, and location of protein expression. Regulation is mediated by cis-acting RNA sequences and structures and transacting RNA binding proteins and non-coding RNAs, including microRNAs. Recent advances in characterization of these regulatory factors have revealed enormous regulatory potential.

This books aims to provide an up-to-date guide to using recombinases in the laboratory. Chapters guide the reader through construction of transgenic animals, recombinase-mediated cassette exchange (RMCE), spatial control of recombinase expression and in vivo delivery approaches, immunohistochemistry and fluorescence-based phenotyping methods, biochemical methods, and recombinase-based production of minimal DNA vectors. 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, Site-Specific Recombinases: Methods and Protocols aims to serve as a valuable resource for working with recombinases, and inspires further creativity in the field.