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.

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.

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.

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 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.

Many exciting discoveries in recent decades have contributed new knowledge to our understanding of the mechanisms that regulate various stages of plant growth and development. Such information, coupled with advances in cell and molecular biology, is fundamental to crop improvement using biotechnological approaches. Two volumes constitute the present work. The ?rst, comprising 22 chapters, commences with introductions relating to gene regulatory models for plant dev- opment and crop improvement, particularly the use of Arabidopsis as a model plant. These chapters are followed by speci?c topics that focus on different developmental aspects associated with vegetative and reproductive phases of the life cycle of a plant. Six chapters discuss vegetative growth and development. Their contents consider topics such as shoot branching, bud dormancy and growth, the devel- ment of roots, nodules and tubers, and senescence. The reproductive phase of plant development is in 14 chapters that present topics such as ?oral organ init- tion and the regulation of ?owering, the development of male and female gametes, pollen germination and tube growth, fertilization, fruit development and ripening, seed development, dormancy, germination, and apomixis. Male sterility and self-incompatibility are also discussed.

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 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

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.

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.

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.

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.

Classical methods for microbial strain engineering, used to improve the production of bioproducts, have serious drawbacks and have been found to be unsuitable for complex strain development applications. In Strain Engineering: Methods and Protocols, powerful new genetic engineering-based strain engineering methods are presented for rational modification of a variety of model organisms. These methods are particularly powerful when utilized to manipulate microbes for which sequenced and annotated genomes are available. Collectively, these methods systematically introduce genome alterations in a precise manner, allowing the creation of novel strains carrying only desired genome alterations. In the first section, E. coli-based bacterial strain engineering strategies are reviewed, while the second section presents analogous microbial engineering strategies for eukaryotic cells using the yeast Saccharomyces cerevisiae as a model. The third section covers examples of the proliferative adaptations of these base technologies to strain engineer industrially important prokaryotic or eukaryotic microbial systems. Written in the highly successful Methods in Molecular Biology (TM) series format, chapters contain 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. Authoritative and accessible, Strain Engineering: Methods and Protocols serves as an ideal guide to scientists in academia, pharmaceutical science, and biotechnology who perform microbial strain engineering.

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.

This book explores the double coding property of DNA, which is manifested in the digital and analog information types as two interdependent codes. This double coding principle can be applied to all living systems, from the level of the individual cell to entire social systems, seen as systems of communication. Further topics discussed include the ubiquitous problem of logical typing, which reflects our inherent incapacity to simultaneously perceive discontinuity and continuity, the problem of time, and the peculiarities of autopoietic living systems. It is shown that the scientific "truths" that appear to be coherent constructions connecting the scientifically verified observations by the rules of logic are in fact always relative and never absolute.

Several years ago, when the discovery of catalytic RNA was recognized in a public manner,many people asked if new ?elds of therapy would soon be available. Although some tentative positive answers were given,nobody would say with certainty that RNA of various kinds was a truly promising means of altering gene expression. In fact,over the past decade,both our knowledge of RNAs with different functions and the utility of RNA in the inhibition or enhancement of gene expression have occurred with great drama. We proceeded in terms of possible therapeutic tools from RNase P and group I introns through "hammerhead" RNA enzymes, antisense technology, and more recently, to RNAi and its derivatives. A useful practical method of RNA delivery in animals will complete the picture. The diversity of RNA and the varied role of it inside cells and in therapy should be a tremendous challenge for young molecular biologists. This volume will make their task easier. Sidney Altman Sterling Professor of Molecular,Cellular & Devel- mental Biology,Nobel Laureate Department of Molecular,Cellular and Developm- tal Biology Yale University V NGTPR 4/23/05 1:00 PM Page VI VI Foreword Delivery of nucleic acids to cells in an animal remains a challenging problem. It is the major obstacle to success of therapeutic approaches using genes and oli- nucleotides,including siRNAs. Solutions found so far by chemists are satisfactory only for transfection of cells in culture.

Genetic Engineering: Principles and Methods presents state-of-the-art discussions in modern genetics and genetic engineering. Recent volumes have covered gene therapy research, genetic mapping, plant science and technology, transport protein biochemistry, and viral vectors in gene therapy, among many other topics. Key features of Volume 27 include: - Identification and Analysis of Micrornas - Dormancy and the Cell Cycle - Long distance peptide and metal transport in plants - Signaling in plant response to temperature and water stresses - Nutrient transport and metabolism in plants - Salt Stress Signaling and Mechanisms of Plant Salt Tolerance - Gene cloning and expression - Assisted folding and assembly of proteins

Agricultural biotechnology takes many forms and applications, with the number and diversity of products ever increasing. With this rapid development, regulatory authorities have sought to keep pace through regulatory adjustments and advances to ensure the safe and beneficial use of this critical technology. The regulatory systems for the U.S. and Canada are not static and must evolve in order to maintain relevance, efficiency and applicability to the challenges encountered. The diverse authors, drawn from the biotechnology industry, academia, government research and regulatory agencies, offer their perspectives of the historical and current system and suggest where it can be improved in the future. Based upon vast experience interacting with the regulatory system, the editors and authors offer demystifying views of the US and Canadian regulatory structures and how they came to be. We know of no other effort to present the biotechnology regulatory systems of the US and Canada in an open forum which will benefit those in the regulated community as well as those charged with oversight of the products of biotechnology, and ultimately the consumer!

Beyond Human is an informative and accessible guide for all those interested in the developing sciences of genetic engineering, bio printing and human cloning. Illustrating the ideas with reference to well-known science fiction films and novels, the author provides a unique insight into and understanding of how genetic manipulation, cloning, and other novel bio-technologies will one day allow us to redesign our species. It also addresses the legitimate concerns about playing God, while at the same time embracing the positive aspects of the scientific trajectory that will lead to our transhuman future."

In 2001 the Human Genome Project succeeded in mapping the DNA of humans. This landmark accomplishment launched the field of genomics, the integrated study of all the genes in the human body and the related biomedical interventions that can be tailored to benefit a person's health. Today genomics, part of a larger movement toward personalized medicine, is poised to revolutionize health care. By cross-referencing an individual's genetic sequence - their genome - against known elements of "Big Data," elements of genomics are already being incorporated on a widespread basis, including prenatal disease screening and targeted cancer treatments. With more innovations soon to arrive at the bedside, the promise of the genomics revolution is limitless. This entry in the What Everyone Needs to Know series offers an authoritative resource on the prospects and realities of genomics and personalized medicine. As this science continues to alter traditional medical paradigms, consumers are faced with additional options and more complicated decisions regarding their health care. This book provides the essential information everyone needs.

Regenerative medicine, encompassing stem cells and tissue engineering, has attracted huge interest within commercial, clinical and government circles, and promises to change medicine itself. This book provides the first detailed examination and critical assessment of the field to be made by social science.

Aspects of genetic engineering research emphasized in this volume are applications to plants (crop plants and grass, both important for human needs) and new methodologies, such as Tar cloning, which make it much easier to isolate specific regions from complex genomes. Another subject discussed is linear DNA replication of prokaryotes.

The newest installment in this superb series presents descriptions of the latest DNA recombinants molecule technology. The text combines reports on basic research in genetics with discussions of specific new industrial applications (as well as refinements of older ones) that are likely to prove highly profitable in the years to come.