This book is open access under a CC BY-NC 2.5 license. This book offers 19 detailed protocols on the use of induced mutations in crop breeding and functional genomics studies, which cover topics including chemical and physical mutagenesis, phenotypic screening methods, traditional TILLING and TILLING by sequencing, doubled haploidy, targeted genome editing, and low-cost methods for the molecular characterization of mutant plants that are suitable for laboratories in developing countries. The collection of protocols equips users with the techniques they need in order to start a program on mutation breeding or functional genomics using both forward and reverse-genetic approaches. Methods are provided for seed and vegetatively propagated crops (e.g. banana, barley, cassava, jatropha, rice) and can be adapted for use in other species.

This book review series presents current trends in modern biotechnology. The aim is to cover all aspects of this interdisciplinary technology where knowledge, methods and expertise are required from chemistry, biochemistry, microbiology, genetics, chemical engineering and computer science. Volumes are organized topically and provide a comprehensive discussion of developments in the respective field over the past 3-5 years. The series also discusses new discoveries and applications. Special volumes are dedicated to selected topics which focus on new biotechnological products and new processes for their synthesis and purification. In general, special volumes are edited by well-known guest editors. The series editor and publisher will however always be pleased to receive suggestions and supplementary information. Manuscripts are accepted in English.

Louis-Marie Houdebine and Jianglin Fan The study of biological functions of proteins and their possible roles in the pathogenesis of human diseases requires more and more relevant animal m- els. Although mice including genetically modified mice offer many possibilities, other non-murine species are absolutely required in some circumstances. Rabbit is one of these species, which has been widely used in biomedical studies. This animal is genetically and physiologically closer to humans including cardiov- cular system and metabolism characteristics. Rabbit is thus more appropriate than mice to study some diseases such as atherosclerosis and lipid metabolism. Because of its larger size, surgery manipulation, bleeding, and turn-over studies are much easier performed in rabbits than in mice. Furthermore, transgenic rabbits can be produced using microinjection and other methods such as lentiviral v- tors. Cloning in rabbits has been proved possible, even though still laborious and time-consuming. Hopefully, functional rabbit ES cell lines will be available in the coming years. Gene deletion or knock-out in rabbits will then become possible.

This new edition explores current and emerging mutagenesis methods focusing specifically on mammalian systems and commonly used model organisms through comprehensive coverage and detailed protocols. Since the first edition, major advances and discoveries have made chromosomal mutagenesis a widely used technique and one that is available to any molecular biology laboratory, and this collection provides detailed protocols, case-studies, and reviews from thought-leaders in the field. 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 fully updated, Chromosomal Mutagenesis, Second Edition aims to help speed scientific discovery and aid in the next advances in the field.

Synthetic Biology is already an object of intensive debate. However, to a great extent the discussion to date has been concerned with fundamental ethical, religious and philosophical questions. By contrast, based on an investigation of the field’s scientific and technological character, this book focuses on new functionalities provided by synthetic biology and explores the associated opportunities and risks. Following an introduction to the subject and a discussion of the most central paradigms and methodologies, the book provides an overview of the structure of this field of science and technology. It informs the reader about the current stage of development, as well as topical problems and potential opportunities in important fields of application. But not only the science itself is in focus. In order to investigate its broader impact, ecological as well as ethical implications will be considered, paving the way for a discussion of responsibilities in the context of a field at a transitional crossroads between basic and applied science. In closing, the requirements for a suitable regulatory framework are discussed. The book is intended as a source of information and orientation for researchers, students and practitioners in the natural sciences and technology assessment; for members of scientific and technological, governmental and funding institutions; and for members of the general public interested in essential information on the current status, prospects and implications of synthetic biology.

DNA replication is arguably the most crucial process at work in living cells. It is the mechanism by which organisms pass their genetic information from one generation to the next and life on Earth would be unthinkable without it. Despite the discovery of DNA structure in the 1950s, the mechanism of its replication remains rather elusive. This work makes important contributions to this line of research. In particular, it addresses two key questions in the area of DNA replication: which evolutionary forces drive the positioning of replication origins in the chromosome and how is the spatial organization of replication factories achieved inside the nucleus of a cell?. A cross-disciplinary approach uniting physics and biology is at the heart of this research. Along with experimental support, statistical physics theory produces optimal origin positions and provides a model for replication fork assembly in yeast. Advances made here can potentially further our understanding of disease mechanisms such as the abnormal replication in cancer.

This book, written by experienced geneticists, covers topics ranging from the natural history of the mouse species, its handling and reproduction in the laboratory, and its classical genetics and cytogenetics, to modern issues including the analysis of the transcriptome, the parental imprinting and X-chromosome inactivation. The strategies for creating all sorts of mutations, either by genetic engineering or by using mutagens, are also reviewed and discussed in detail. Finally, a last chapter outlines the methodology used for the analysis of complex or quantitative traits. The authors also discuss the importance of accurate phenotyping, which is now performed in the mouse clinics established worldwide and identify the limits of the mouse model, which under certain circumstances can fail to present the phenotype expected from the cognate condition in the human model. For each chapter an up-to-date list of pertinent references is provided. In short, this book offers an essential resource for all scientists who use or plan to use mice in their research.

The rapid progression of genetics and molecular biology has turned chromosomal engineering from science fiction to reality, with the successful production of transgenic animals with engineered chromosomes and chromosomes developed for pharmaceutical protein production which are now ready for the medical industry. Mammalian Chromosome Engineering: Methods and Protocols provides the reader with up-to date information on this rapidly evolving field and strives to take the reader into the exciting realm of chromosomal engineering from the basic principles to the practical applications of these new technologies. The five overview and ten protocol chapters cover the engineering of chromosomes with extrachromosomal vectors and transposon systems, the manipulation of naturally occurred minichromosomes, the generation and engineering of synthetic artificial chromosomes, and the induced de novo platform artificial chromosome system. Written in the highly successful Methods in Molecular Biology (TM) series format, protocols chapters contain brief 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, Mammalian Chromosome Engineering: Methods and Protocols serves as a bench-side resource for current protocols and aims to help scientists to explore the many prospects for future research and vital applications.

With the rapid proliferation of RNAi applications in basic and clinical sciences, the challenge has now become understanding how components of RNAi machinery function together in a regulated manner. Argonaute proteins are the central effectors of RNAi and are highly conserved among eukaryotes and some archaebacteria. These RNA-binding proteins use small guide RNAs to silence expression of genes at the mRNA and DNA levels. In Argonaute Proteins: Methods and Protocols, expert researchers in this burgeoning field provide detailed, up-to-date methods to study Argonaute protein functions and interactions in a wide variety of cell types ranging from yeast to mammalian systems, as well as in vitro. Written in the highly successful Methods in Molecular Biology (TM) series format, chapters include brief introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Practical and authoritative, Argonaute Proteins: Methods and Protocols serves as a vital reference for both experienced and novice scientists approaching the vast complexities of RNAi research.

Central to the synthesis of proteins, the performance of catalysis, and many other physiological processes, the aberrant expression of which can be linked to human diseases including cancers, RNA has proven to be key target for therapeutics as well as a tool for therapy. In RNA Therapeutics: Function, Design, and Delivery, expert contributors from a broad spectrum of scientific backgrounds highlight the roles that messenger RNAs and small RNAs can play in biology and medicine. While covering the five major RNA-based drugs, namely the use of ribozymes to cleave and/or correct mRNA transcript, the use of siRNA for targeted silencing of gene transcripts, the use of aptamers, like short RNA molecules, for neutralizing the protein functions, the use mRNA-transfected DCs to activate immune system against tumor cells, as well as the use of RNA to reprogram T and/or DC cell function, this extensive volume brings together the fields of coding (mRNA) and non-coding RNA such as ribozymes, RNAse P, siRNAs, and miRNAs into one convenient source. Written in the highly successful Methods in Molecular Biology (TM) series format, the cutting-edge protocol chapters contain introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and practical tips on troubleshooting and avoiding known pitfalls. Also, the book contains several excellent reviews for teaching purposes. Authoritative and comprehensive, RNA Therapeutics: Function, Design, and Delivery provides key models and tools which will assist researchers in increasing our understanding of RNA functions, modifications, and their involvement in diseases in order to lead to the design of vital new RNA-based therapeutics.

With an increasing human population and a decreasing amount of arable land, creative improvements in agriculture will be a necessity in the coming decades to maintain or improve the standard of living. In Plant Chromosome Engineering: Methods and Protocols, expert researchers present techniques for the modification of crops and other plant species in order to achieve the goal of developing the much needed novel approaches to the production of food, feed, fuel, fiber, and pharmaceuticals. This volume examines vital topics such as transformation procedures, chromosome painting, production of engineered minichromosomes, gene targeting and mutagenesis, site specific integration, gene silencing, protein expression, chromosome sorting and analysis, protocols for generating chromosomal rearrangements, enhancer trapping, and means of studying chromosomes in vivo. As a part of the highly successful Methods in Molecular Biology (TM) series, the methodological chapters include brief introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and professional tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Plant Chromosome Engineering: Methods and Protocols highlights the spectrum of tools currently available for modifying plant genomes and chromosomes and provides the foundation for crucial future developments.

This volume offers an up-to-date overview of biotechnologically oriented barley research. It is structured into two major sections: the first focusing on current agricultural challenges and approaches to barley improvement, and the second providing insights into recent advances in methodology. Leading scientists highlight topics such as: the global importance of barley; genetic diversity and genebanks; domestication; shoot and inflorescence architecture; reproductive development; mineral nutrition; photosynthesis and leaf senescence; grain development; drought tolerance; viral and fungal pathogens; phytophagous arthropods; molecular farming; sequence resources; induced genetic variation and TILLING; meiotic recombination; Hordeum bulbosum; genome-wide association scans; genomic selection; haploid technology; genetic engineering; and whole plant phenomics. Providing comprehensive information on topics ranging from fundamental aspects to specific applications, this book offers a useful resource for scientists, plant breeders, teachers and advanced students in the fields of molecular and plant cell biology, plant biotechnology, and agronomy.

In the post-genomic era, in vitro mutagenesis has emerged as a critically important tool for establishing the functions of components of the proteome. The third edition of In Vitro Mutagenesis Protocols represents a practical toolbox containing protocols vital to advancing our understanding of the connection between nucleotide sequence and sequence function. Fully updated from the previous editions, this volume contains a variety of specialty tools successfully employed to unravel the intricacies of protein-protein interaction, protein structure-function, protein regulation of biological processes, and protein activity, as well as a novel section on mutagenesis methods for unique microbes as a guide to the generalization of mutagenesis strategies for a host of microbial systems. Written in the highly successful Methods in Molecular Biology (TM) series format, chapters include brief introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and expert tips on troubleshooting and avoiding known pitfalls. Authoritative and up-to-date, In Vitro Mutagenesis Protocols, Third Edition offers today's researchers a valuable compendium of reliable and powerful techniques with which to illuminate the proteome and its rich web of biological implications.

Biolistic transfection represents a direct physical gene transfer approach in which nucleic acids are precipitated on biologically inert high-density microparticles (usually gold or tungsten) and delivered directly through cell walls and/or membranes into the nucleus of target cells by high-velocity acceleration using a ballistic device such as the gene gun. Biolistic DNA Delivery: Methods and Protocols provides a comprehensive collection of detailed protocols intended to provide the definitive practical guide for the novice as well as for the advanced gene transfer expert on how to introduce nucleic acids into eukaryotic cells using the biolistic technique. Split into six convenient sections, this detailed volume covers biolistic gene transfer into plants, nematodes, and mammalian cells, both in vitro and in vivo, as well as the use of gene gun-mediated DNA vaccination in various experimental animal models of human diseases, and the description of biolistic delivery of molecules other than nucleic acids. Written in the highly successful Methods in Molecular Biology (TM) 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. All-inclusive and cutting-edge, Biolistic DNA Delivery: Methods and Protocols brings together the knowledge and the experience of leading experts in the field of gene transfer in order to serve all researchers who wish to further our abilities in this vital field.

Nuclear Reprogramming: Methods and Protocols, Second Edition includes not only classic methods to perform nuclear transfer in different species but also several techniques to assess the early and late development of the reconstructed embryos, at the cellular, molecular, and epigenetic level. Over the past several years, many technical improvements have been made to improve somatic cell nuclear transfer (SCNT) efficiency, all of which are reflected in the detailed chapters of this fully revised collection. 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 up-to-date, Nuclear Reprogramming: Methods and Protocols, Second Edition will be of interest not only to cloners but also to researchers concerned with studying the development of embryos.

Since the publication of the first edition, lentivirus vector-based technologies, through in vitro and in vivo gene transfer in eukaryotic animal cells, continue to offer the most promising opportunities for curing genetic disorders, as well as cancer and infectious diseases. Lentivirus Gene Engineering Protocols, Second Edition reflects the spectacular progress made in the field with a set of cutting-edge methods contributed by highly respected scientists. Beginning with a thorough overview of the most recent lentivirus developments, the book continues with detailed protocols including sections on the advances in lentiviral vector technology, new lentiviral vector applications, involving transgenic human embryonic stem cells and fetal gene therapy among other topics, as well as the invaluable breakthroughs in LV-mediated expression of microRNAs. Written in the highly successful Methods in Molecular Biology (TM) series format, chapters include introductions to their respective subjects, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes sections, highlighting tips on troubleshooting and avoiding known pitfalls. Authoritative and timely, Lentivirus Gene Engineering Protocols, Second Edition covers the most relevant issues and techniques of LV-based gene engineering, thus representing a complete theoretical and practical guide for scientists still unfamiliar with LV technologies and those who simply wish to know more about this vital area of study.

Conceived with the aim of sorting fact from fiction over genetically modified (GM) crops, this book brings together the knowledge of 30 specialists in the field of transgenic plants. It covers the generation and detection of these plants as well as the genetic traits conferred on transgenic plants. In addition, the book looks at a wide variety of crops, ornamental plants and tree species that are subject to genetic modifications, assessing the risks involved in genetic modification as well as the potential economic benefits of the technology in specific cases. The book's structure, with fully cross-referenced chapters, gives readers a quick access to specific topics, whether that is comprehensive data on particular species of ornamentals, or coverage of the socioeconomic implications of GM technology. With an increasing demand for bioenergy, and the necessary higher yields relying on wider genetic variation, this book supplies all the technical details required to move forward to a new era in agriculture.

The generation of genetically modified mice is absolutely crucial to gene function studies today, primarily because mice are genetically similar to man and because gene function studies in mice are in the context of a whole organism, making them particularly useful. In Transgenic Mouse Methods and Protocols, Second Edition, expert research explore current advances in the field through detailed laboratory protocols. Chapters provide a general introduction outlining how to deal with mice and how to generate transgenic mouse models, explore the generation of conditional and induced knockout and transgenic mice, and offer alternative routes to studying gene function in mice. Composed in the highly successful Methods in Molecular Biology (TM) series format, each chapter contains a brief introduction, step-by-step methods, a list of necessary materials, and a Notes section which shares tips on troubleshooting and avoiding known pitfalls. Comprehensive and state of the art, Transgenic Mouse Methods and Protocols, second Edition is the ideal guide for all researchers interested in the latest information about the production and analysis of transgenic and knockout mice.

The de novo fabrication of custom DNA molecules is a transformative technology that significantly affects the biotechnology industry. Basic genetic engineering techniques for manipulating DNA in vitro opened an incredible field of opportunity in the life sciences. In, Gene Synthesis: Methods and Protocols expert researchers in the field detail many of the methods which are now commonly used to fabricate DNA . These include methods and techniques for the assembly of oligonucleotide, cloning of synthons into larger fragments, protocols and software applications, and educational and biosecurity impacts of gene synthesis. Written in the highly successful Methods in Molecular Biology (TM) series format, the chapters include the kind of detailed description and implementation advice that is crucial for getting optimal results in the laboratory. Thorough and intuitive, Gene Synthese: Methods and Protocols aids scientists in understanding all the different stages of a complex gene synthesis process, while refining their understanding of gene synthesis and determine what part of the process they can or should do in their laboratory and what parts should be contracted to a specialized service provider.

Bacterial genomics is a mature research interdisciplinary field, which is approached by ecologists, geneticists, bacteriologists, molecular biologists and evolutionary biologists working in medical, industrial and basic science. Thanks to the large diffusion of bacterial genome analysis, Bacterial Pangenomics: Methods and Protocols is able to provide the most recent methodologies about the study of bacterial pangenomes by covering the three major areas: the experimental methods for approaching bacterial pangenomics, the bio informatic pipelines for analysis and annotation of sequence data and finally the methods for inferring functional and evolutionary features from the pangenome. Written in the 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 protocols and notes on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, Bacterial Pangenomics: Methods and Protocols will serve as a field guide for both qualified bacterial genomics investigators who want to update their technical knowledge, for less experienced researchers who want to start working with bacterial genomics and pangenomics, as well as serving as a manual and supplemental textbook for graduate students of genomics and bioinformatics.

Directed Evolution Library Creation: Methods and Protocols, Second Edition presents user-friendly protocols for both proven strategies and cutting-edge approaches for the creation of mutant gene libraries for directed evolution. As well as experimental methods, information on current computational approaches is provided in a user-friendly format that will allow researchers to make informed choices without needing to comprehend the full technical details of each algorithm. Directed evolution has become a fundamental approach for engineering proteins to enhance activity and explore structure-function relationships, and has supported the rapid development of the field of synthetic biology over the last decade. Divided into three convenient sections, topics include point mutagenesis strategies, recombinatorial methods wherein genetic diversity is sourced from multiple parental genes that are combined via either homology-dependent or -independent techniques and a variety of computational methods to guide the design and analysis of mutant libraries. Written in the 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 protocols and notes on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, Directed Evolution Library Creation: Methods and Protocols, Second Edition will serve as a reliable manual for both novice and experienced protein engineers and synthetic biologists and will enable further technical innovation and the exploitation of directed evolution for a deeper understanding of protein design and function.

DNA Vaccines: Methods and Protocols, Third Edition explores innovative approaches and technologies used to design, deliver, and enhance the efficacy of DNA vaccines. Featuring applications which should be of great value in moving vaccines from research to clinic, this detailed volume includes sections on DNA vaccine design and enhancement, delivery systems, production, purification, and quality, as well as chapters on new vaccine applications. Written in the highly successful Methods in Molecular Biology series format, chapters contain introductions 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 practical, DNA Vaccines: Methods and Protocols, Third Edition serves the important role of further documenting the potential of the DNA vaccination as a platform technology for treatment and prevention of human disease.

This book provides a comprehensive, up-to-date overview of the opportunities and challenges of the complex field of synthetic biology, which combines various scientific disciplines. The emerging field of synthetic biology employs biotechnological approaches to recreate and enhance basic biological structures, intracellular processes and whole organisms. The book addresses a broad range of topics, including redesigning complex metabolic pathways, DNA/RNA and protein engineering, as well as novel synthetic biomaterials. It discusses both "bottom up" and "top down" approaches and presents the latest genome engineering tools with predictions about how these could change our way of thinking and working. Since the use of synthetic biology raises a number of ethical questions, a chapter is devoted to public awareness and risk management.The book is of interest to scientists from both academia and industry, as well as PhD students and postdocs working in the field

Among the many types of DNA binding domains, C2H2 zinc finger proteins (ZFPs) have proven to be the most malleable for creating custom DNA-binding proteins. In Engineered Zinc Finger Proteins: Methods and Protocols, expert researchers from some of the most active laboratories in this field present detailed methods, guidance, and perspectives. The volume contains sections covering the engineering of ZFPs, methods for the creation, evaluation, and delivery of artificial transcription factors (ATFs), methods for the creation and evaluation of zinc finger nucleases (ZFNs), and a collection of the several applications and assays beyond ATFs and ZFNs, including zinc finger transposases and ChIP-seq methodology amongst other subjects. Written in the highly successful Methods in Molecular Biology (TM) series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes on troubleshooting and avoiding known pitfalls. Comprehensive and cutting-edge, Engineered Zinc Finger Proteins: Methods and Protocols aims to aid both seasoned practitioners and new investigators with its vital methods and insights as they seek to create the next generation of engineered ZFPs and applications.

Homing Endonucleases: Methods and Protocols aims at providing molecular biologists with a comprehensive resource to identify and characterize homing endonucleases from genomic sequence, to deduce the biological basis of binding and cleavage specificity, as well as to provide protocols to redesign endonuclease target specificity for genome-editing applications. Engineering of designer homing endonucleases has set the stage for genome editing of complex eukaryotic genomes with a broad range of potential applications including targeted gene knockouts in model organisms and gene therapy in humans, making this book a valuable resource for future research. 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 practical, Homing Endonucleases: Methods and Protocols serves as a key reference for all labs studying site-specific DNA endonucleases.