A collection of standard and cutting-edge techniques for using Xenopus oocytes and oocytes/egg extracts to reconstitute biological and cellular processes. These readily reproducible methods take advantage of the oocyte's impressive protein abundance, its striking protein translation capacity, and its breathtaking possibilities for the assembly of infectious viral particles by single cell injection of multiple RNAs. The authors focus on the versatility of frog oocytes and egg extracts in cell biology and signal transduction, and cover all the major uses of oocytes/extracts as experimental models.

During the 20th century Xenopus has flourished as an accessible experimental model system for studying vertebrate biology. The second edition of Xenopus Protocols:Post-Genomic Approaches expands upon the previous edition with novel approaches inspired by Xenopus tropicalis and the sequencing of its genome. Xenopus tropicalis genomics and tractable genetics combined with the unique experimental advantages of Xenopus laevis and their distinctive phylogenetic position make them unique model systems for vertebrate embryonic development in the 21st century. Written in the highly successful Methods in Molecular BiologyTM series format, chapters include 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. Authoritative and practical, Xenopus Protocols: Post-Genomic Approaches, Second Edition provides a toolkit that will enable every laboratory to maximize the power of this extraordinary experimental system in the post-genomic age.

Amphibian embryos are supremely valuable in studies of early vertebrate development because they are large, handle easily, and can be obtained at many interesting stages. And of all the amphibians available for study, the most valuable is Xenopus laevis,which is easy to keep and ovulates at any time of year in response to simple hormone injections. Xenopusembryos have been studied for years but this is a particularly exciting time for the field. Techniques have become available very recently that permit a previously impossible degree of manipulation of gene expression in intact embryos, as well as the ability to visualize the results of such manipulation. As a result, a sophisticated new understanding of Xenopusdevelopment has emerged, which ensures the species’ continued prominent position among the organisms favored for biological investigation. This manual contains a comprehensive collection of protocols for the study of early development in Xenopusembryos. It is written by several of the field’s most prominent investigators in the light of the experience they gained as instructors in an intensive laboratory course taught at Cold Spring Harbor Laboratory since 1991. As a result it contains pointers, hints, and other technical knowledge not readily available elsewhere. This volume is essential reading for all investigators interested in the developmental and cell biology of Xenopusand vertebrates generally. Many of the techniques described here are illustrated in an accompanying set of videotapeswhich are cross-referenced to the appropriate section of the manual.

The process whereby a single cell, the fertilized egg, develops into an adult has fascinated for centuries. Great progress in understanding that process, h- ever, has been made in the last two decades, when the techniques of molecular biology have become available to developmental biologists. By applying these techniques, the exact nature of many of the interactions responsible for forming the body pattern are now being revealed in detail. Such studies are a large, and it seems ever-expanding, part of most life-science groups. It is at newcomers to this field that this book is primarily aimed. A number of different plants and animals serve as common model org- isms for developmental studies. In Molecular Methods in Developmental Bi- ogy: Xenopus and Zebrafish, a range of the molecular methods applicable to two of these organisms are described, these are the South African clawed frog, Xenopus laevis, and the zebrafish, Brachydanio rerio. The embryos of both of these species develop rapidly and externally, making them particularly suited to investigations of early vertebrate development. However, both Xenopus and zebrafish have their own advantages and disadvantages. Xenopus have large, robust embryos that can be manipulated surgically with ease, but their pseudotetraploidy and long generation time make them unsuitable candidates for genetics. This disadvantage may soon be overcome by using the diploid Xenopus tropicalis, and early experiments are already underway. The transp- ent embryos of zebrafish render them well-suited for in situ hybridization and immunohistochemistry, and good for observing mutations in genetic screens.

本书的实验方案涵盖了爪蟾胚胎、成体的形态发生,爪蟾中基因表达操作,胚胎的获取和实验常规操作等内容。

Nuclear Transfer Protocols: Cell Reprogramming and Transgenesis is a comprehensive review of nuclear transfer technology in vertebrates, aimed at reprogramming differentiated nuclei and effecting targeted gene transfer. The emphasis here is on providing readily reproducible techniques for the gene- tion of cloned embryos and animals in a number of key research and commercially important vertebrates. Additional chapters provide alternative cutting-edge methods for nuclear transfer, such as zona-free nuclear transfer and serial nuclear transfer. Of immense practical benefit are descriptions of procedures associated with cloning, such as in vitro maturation of oocytes, activation and culture of cloned embryos, maintenance of pregnancy, and neonatal care of clones. Nuclear Transfer Protocols: Cell Reprogramming and Transgenesis also provides an understanding of the factors involved in nuclear reprogramming, which is imperative for the success of cloning. A section dealing with such cloning-related issues as aging and normality of clones is also included making this an essential comprehensive handbook for research and commercial labo- tories involved in, or intending to work on, nuclear transfer. The volume will prove beneficial to molecular biologists, stem cell biologists, clinicians, biotechnologists, students, veterinarians, and animal care technicians involved with reprogramming, nuclear transfer, and transgenesis.

Developmental biology is one of the most exciting and fast-growing fields today. In part, this is so because the subject matter deals with the innately fascinating biological events—changes in form, structure, and function of the org- ism. The other reason for much of the excitement in developmental biology is that the field has truly become the unifying melting pot of biology, and provides a framework that integrates anatomy, physiology, genetics, biochemistry, and cellular and mole- lar biology, as well as evolutionary biology. No longer is the study of embryonic development merely “embryology.” In fact, development biology has produced - portant paradigms for both basic and clinical biomedical sciences alike. Although modern developmental biology has its roots in “experimental emb- ology” and the even more classical “chemical embryology,” the recent explosive and remarkable advances in developmental biology are critically linked to the advent of the “cellular and molecular biology revolution.” The impressive arsenal of expe- mental and analytical tools derived from cell and molecular biology, which promise to continue to expand, together with the exponentially developing sophistication in fu- tional imaging and information technologies, guarantee that the study of the devel- ing embryo will contribute one of the most captivating areas of biological research in the next millennium.

More than 40 years after the discovery of the nucleosome as the fun- mental unit of chromatin, the multifaceted problem of how variations in ch- matin structure affect the activity of the eukaryotic genome has not been solved. However, during the past few years research on chromatin structure and fu- tion has gained considerable momentum, and impressive progress has been made at the level of concept development as well as filling in crucial detail. The structure of the nucleosome has been visualized at unprecedented reso- tion. Powerful multisubunit enzymes have been identified that alter histone/ DNA interactions in ways that expose regulatory sequences to factors initi- ing and regulating such nuclear processes as transcription. Though the imp- tance of posttranslational modifications of histones, notably their acetylation, has long been known, the finding that a number of bona fide regulators increase transcription by acetylating nucleosomes has lent new support to the old idea that the process of gene regulation is intimately related to the nature of the chromatin environment. A wealth of nonhistone proteins contribute to a continuum of structures with distinct biochemical properties and varying degrees of DNA condensation. Perhaps the most important conclusion from a large number of studies is a fresh appreciation of the dynamic nature of chromatin structure, the built-in flexibility providing the basis for regulation.

Even though Xenopus is one of the two most popular non-mammalian animals used in biomedical research, its value in the lab suffers from a lack of standardization regarding their optimal care, breeding, and housing. Filling the need for such a reference, The Laboratory Xenopus sp provides researchers and lab managers with a practical, step-by-step m

This volume explores techniques used to study and experiment with Xenopus in order to model or understand human disease. The chapters in this book cover topics such as implementation of CRISPR/Cas9 and TALEN in Xenopus from the design stage up to the genotyping stage; methods to explore generation of knock-in animals; cancer modelling and in vivo screening of congenital heart disease; electroporation; and phenotyping at the organismal level, cellular level, and proteome level. 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. Cutting-edge and comprehensive, Xenopus: Methods and Protocols is a valuable resource for researchers who are interested in expanding their knowledge of this wide and developing field. This book will aid in keeping Xenopus at the forefront for biochemical, cell biological, and developmental studies and forward it as a preclinical disease model.