Advanced Protocols For Animal Transgenesis

This laboratory manual, published in cooperation with the International Society for Transgenic Technology (ISTT), provides almost all current methods that can be applied to the creation and analysis of genetically modified animals. The chapters have been contributed by leading scientists who are actively using the technology in their laboratories. Based on their first-hand experience the authors also provide helpful notes and troubleshooting sections. Topics range from standard techniques, such as pronuclear microinjection of DNA, to more sophisticated and modern methods, such as the derivation and establishment of embryonic stem (ES) cell lines, with defined inhibitors in cell culture medium. In addition, related topics with relevance to the field are addressed, including global web-based resources, legal issues, colony management, shipment of mice and embryos, and the three R’s: refinement, reduction and replacement.

This laboratory manual, published in cooperation with the International Society for Transgenic Technology (ISTT), provides almost all current methods that can be applied to the creation and analysis of genetically modified animals. The chapters have been contributed by leading scientists who are actively using the technology in their laboratories. Based on their first-hand experience the authors also provide helpful notes and troubleshooting sections. Topics range from standard techniques, such as pronuclear microinjection of DNA, to more sophisticated and modern methods, such as the derivation and establishment of embryonic stem (ES) cell lines, with defined inhibitors in cell culture medium. In addition, related topics with relevance to the field are addressed, including global web-based resources, legal issues, colony management, shipment of mice and embryos, and the three R’s: refinement, reduction and replacement.

The past decade has witnessed a spectacular explosion in both the devel- ment and use of transgenic technologies. Not only have these been used to aid our fundamental understanding of biologic mechanisms, but they have also faci- tated the development of a range of disease models that are now truly beginning to impact upon our approach to human disease. Some of the most exciting model systems relate to neurodegenerative disease and cancer, where the availability of appropriate models is at last allowing radically new therapies to be developed and tested. This latter point is of particular significance given the current concerns of the wider public over both the use of animal models and the merits of using genetically modified organisms. Arguably, advances of the greatest significance have been made using mammalian systems—driven by the advent of embryonic stem-cell–based strategies and, more recently, by cloning through nuclear transfer. For this reason, this new edition of Transgenesis Techniques focuses much more heavily on manipulation of the mammalian genome, both in the general discussions and in the provision of specific protocols.

A transgenic animal is one that is genetically modified to carry genes from another species. Transgenic species can be raised to carry potentially useful genes from a variety of species. While the topics of genetic engineering and cloning are controversial, the reality is that these technologies offer tremendous benefits to society - from offering a framework for developing and screening medical therapies, to enhancing the safety and nutrition of the food we eat. One potential application of research into transgenic animal technology is the creation of domestic animals genetically designed to express a certain human disease and therefore serve as models for the study and treatment of human illnesses. Although many mouse models of human diseases are available today, such models in large domestic animals physiologically more similar to humans are sparse and critically needed. Further research in this field will undoubtedly uncover many more direct and indirect benefits of this technology. Transgenic animal technologies and the ability to introduce functional genes into animals have revolutionized our ability to address complex biomedical and biological questions. This well-illustrated handbook covers the technical aspects of gene transfer - from molecular methods to whole animal considerations - for important laboratory and domestic animal species. It describes methodologies as employed by leading laboratories and is a key resource for researchers, as well as a tool for training technicians and students. This second edition incorporates updates on a variety of genetic engineering technologies ranging from microinjection and ES cell transfer to nuclear transfer in a broad range of animal modeling systems. Contains a comprehensive collection of transgenic animal and gene transfer methods Discusses background and introduction to techniques and animal systems Teaches practical step-by-step protocols New section on analysis

This revised and updated edition of a much-acclaimed classic presents a collection of readily reproducible techniques for the generation and analysis of transgenic animals, with emphasis on the manipulation of the mammalian genome. It concentrates on the creation and maintenance of genetically modified murine strains, providing access to the germline by both conventional pronuclear injection, and by retroviral and adenoviral infection. Extensive coverage is also given to the generation, maintenance, and manipulation of embryonic stem cell lineages, with protocols for both constitutive and conditional (Cre-lox or the Flp-frt systems) gene targeting. Additional chapters provide cutting-edge techniques for the cryopreservation of both male and female germlines, for the generation of transgenic sheep by nuclear transfer, and for the basic analysis of transgenic organisms.

The technique of microinjection is used to tranfer biological materials - RNA, proteins, oligonucleotides and especially cloned genes - into cultured somatic cells or embryos. Transgenic cells or animals - mice, rabbits or insects - allow the study of a wide range of cellular processes, such as gene expression, signal transduction or the cytoskeleton. More than 30 protocols on microinjection experiments and transgenesis studies developed in renowned laboratories are described in this manual. Special topics are the generation of transgenic mice using YAC, ligand-dependent site-specific recombination in embryonic stem cells, morula aggregation to generate germline chimeras as well as the analysis of injected cells.

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 BiologyTM 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.